PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein

PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein Rui Li, Zeyu Qi, Yueyang Wanghuang, Dechuan Wu, Yonghan Xu, Wei Tang, and 11 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7640593/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Sequence replacement is the most direct and powerful genome editing strategy. However, it remains currently an urgent need to develop protospacer adjacent motif (PAM)-less, off-target-free and simple tools for precise sequence replacement. To address this challenge, we fused a rice-derived AT-rich pincer-like elements (APE), which is composed of unique repeat-spacer array, to donor template against target sequence, thus forming guider and donor template (gdt). The donor template harbors multiple sites of DNA fragment insertion/deletion (MsDFID) which function as donor sites. APE plays as MsDFID gdt scaffold to repurpose Cas3 or Cas9 to mediate transposition of DNA fragment insertion/deletion from MsDFID donor template into genome target in E . coli , thus realizing seamless sequence replacement. These results established putative gdt/Cas3 or gdt/Cas9 ribonucleoprotein as compact genome editors which feature PAM-lessness, no observable off-target, and simplicity based on the dual role of MsDFID gdt per se as both guider and donor template. This strategy provides significant potential for precise sequence replacement in both animals and plants. AT-rich pincer-like elements (APE) gdt/Cas3 or gdt/Cas9 ribonucleoprotein genome editing sequence replacement Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction For CRISPR-Cas9 systems, it is possible to achieve precise integration of new DNA following Cas9 cleavage either through homologous recombination (HR) (Yu et al., 2019 ; Jasin et al., 2013) or error-prone non-homologous end joining (NHEJ) (Schmid-Burgk et al., 2016 ; Suzuki et al., 2016 ). However, requirement of the recognition of protospacer adjacent motif (PAM) limits target site recognition to a subset of sequences in DNA manipulation by CRISPR-Cas enzymes. To overcome this constraint, Walton et al ( 2020 ) engineered specific variant of Streptococcus pyogenes Cas9 (SpCas9) that could target almost all PAMs, thus eliminating NGG PAM requirement. Strecker et al reported Tn7-like transposases subunit(s) from cyanobacteria can be reprogrammed through association with type V-K CRISPR effector (Cas12k) to insert DNA into E. coli genome (Strecker et al., 2019 ). The reconstitution strategy can achieve unidirectional insertion of DNA segment of up to 2.5 kb into unique sites downstream of protospacer with frequencies of up to 80% but without positive selection. Nevertheless, this does not belong to means of precise sequence replacement. Yu et al ( 2019 ) described a strategy which realized gene knock-in rate of up to 65/40% for 0.7/2.5 kilobase inserts, at various genomic loci in human cancer and stem cells, respectively, via using Cas9 and 5′-modified double-stranded DNA as donor template. Similarly, Lu et al ( 2020 ) employed this strategy to insert sequences of up to 2.049 kilobase pairs into rice genome at efficiency of 25%. However, the two cases of sequence replacement relied on specificly designed schemes and sophisticated operations with the limitation of PAM and risk of off-target. Prime editors are CRISPR-Cas9 nickase (H840A)-reverse transcriptase accompanied with prime editing guide RNAs and can generate base conversions, and small insertions and deletion in plant and animal cells without donor DNA or double-strand breaks (Anzalone et al., 2019 ; Lin et al., 2020 ; Lin et al., 2021 ). Further, Sun et al ( 2024 ) developed a third-generation PrimeRoot editors which employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genome. But, all of these strategies face issues of PAM requirement and off-target. So, precise, PAMless and off-target-free sequence replacement still remains practically challenging. Here, via fusing donor sequence targeting genome site and a rice-derived AT-rich pincer-like elements (APE), we realized the precise sequence replacement in E . coli . In this system, APE establishes a dual role of the donor sequence as both guider and donor template (gdt). The knock-out experiment of Cas genes cas3 , or cas8 and cas11 , or cas11 , cas7 and cas5 , or cas5 and cas6 , in a streamlining way, confirmed that, among E . coli CRISPR-Cas3 system, Cas3 might be the only Cas protein required for the APE-mediated targeted sequence replacement in E . coli . Meanwhile, gdt-APE fusion can also directed Cas9 to achieve precise sequence replacement in cas3 -knockout E . coli strain. This simplifies the tool from three components (an enzyme, guide RNA and donor DNA/RNA) to just a cutting enzyme, E . coli Cas3 or Cas9, and gdt. This simplicity in combination with minimal off-target and no PAM requirement will probably make the tool particularly advantageous for easier vector-based delivery into cells, a wider range of targetable genomic sequences and more purposes of genome editing. We thus conclude that endogenous Cas3 can be programmed to achieve precise sequence replacement in E . coli . 2. Results 2.1 . Structure of AT-rich pincer-like elements (APE) from rice We found that the promoter region of rice gene Ideal Plant Architecture1 ( IPA1 ) harbors a kind of unique structure, AT-rich pincer-like elements (hereafter named APE) (526-bp in length, -1190 to -1715 bp from start code ATG) (Fig. 1 A). The APE consists of 8 direct and 9 reverse repeats of 13 bp which are interspaced by short DNA fragments of 16–19 bp (Fig. 1 B). In fact, both the 8 direct repeats and spacers are basically complementary to their respective reverse repeats and spacers, respectively, in the APE (Fig. 1 B). There are only 9 single nucleotide polymorphisms (SNPs) across all of these 17 repeats (Fig. 1 B). So, this sequence, with A/T bases as high as 74%, is predicted to be able to form a symmetric hairpin structure with several large or small loops in two stems (Fig. S1 ). In fact, APEs were predicated to form various folding patterns at RNA secondary structure level (Fig. S1 ). 2.2 . Fusion of MsDFID guide and donor template (gdt) and APE primes precise sequence replacement in E. coli Although no obvious target of APE spacers was found, we still wonder whether APEs can mediate genome editing. To test the hypothesis, we synthesized a DNA sequence which is designed as donor template against target gene aroA (3-phosphoshikimate 1-carboxyvinyltransferase, NP_309018.1), a functional gene of E. coli . Compared with its target region of an around 350 bp in aroA , the synthesized sequence contains multi-sites (6 sites) with DNA fragment insertion/deletion (MsDFID) (Fig. 2 A and Table S2). These 6 sites act as donor sites, among which 5 and 1 sites harbor DNA fragment insertion (9 bp, 15 bp, 21 bp, 30 bp and 40 bp, in length, respectively) and 12 bp deletion, respectively (Fig. 2 A and Table S2). Then the MsDFID sequence was fused with APE, thus forming MsDFID donor template-APE fusion which was ligated into the downstream of lacZ promoter in modified T-vector pUC57 (hereafter named pUC57m) to yield vector F7869 (Fig. 2 A). So, F7869, which possesses MsDFID donor template, might be a donor vector. Most reviews on genetic recombination support the statement that, in bacteria, all of homology-directed DNA repair (HDR) pathways are primarily dependent on RecA protein (Dutra et al., 2007 ). So, to rule out the possibility of endogenous RecA-dependent HDR, we selected E . coli strain including TOP10 or T1 which bears loss-of-function recA gene for investigating sequence replacement event thereafter in this study. Meanwhile, RecA-independent recombination, which relies upon the knockout of exonucleases including ExoI, ExoVII, ExoX, and RecJ, was also ruled out, because these exonucleases is efficient in these two strains (Dutra et al., 2007 ). To check whether MsDFID donor template-APE fusion mediates sequence replacement in the target region in E . coli genome, we isolated the genomic DNA of F7869-transformed E . coli . Desired sequence replacement (DNA fragment insertion or deletion) in target region is expected to produce larger or smaller amplicons. We then performed genotyping PCR with genomic DNA of transformed E. coli . Insertion or deletion frequency was also quantitated for all the 6 target sites. To exclude the contamination of plasmid DNA as PCR template, the used primer set, FF2 and FR4, were designed to be specific for the genomic sequences of aroA flanking the homologous arms, thus ensuring genotyping PCR (namely flanking PCR) product to be E. coli genome-specific but not for the donor vector (Fig. 2 A). The resulting genotyping PCR product was Sanger-sequenced and the chromatogram results showed that the clear double peaks appeared just corresponding to the targeting position of No.1 donor site with a DNA insertion in the MsDFID donor template of vector F7869 (Fig. 2 B). The double peaks demonstrated that the PCR amplicons were heterozygous and consists of wild-type and edited sequences in genomic target region, therefore indicating that some genomic DNAs had been successfully edited (Fig. 2 B). Next, the total genotyping PCR amplicons were ligated into T-vector and the resulting plasmids were used to transform E. coli . The 40 single E. coli clones were collected to extract the recombination plasmids for Sanger-sequencing inserted PCR amplicons. Sequencing result identified two types of PCR amplicons which defined two independent sequence replacement events between No.1 and No. 2 donor sites in MsDFID donor template and their respective target sites in aroA , respectively (Fig. 2 B). Sequence replacement rates were measured up to 22.5% and 2.5% for the two cases, respectively (Fig. 2 B). Clearly, the replacement efficiency in No.1 donor site is much higher than that in No.2, and thus inversely proportional to the distance of the respective donor site of MsDFID donor template to APE in donor vector. Meanwhile, possibly due to limited number of selected E. coli single clones for sequencing inserted PCR amplicons, we failed to identify the sequence replacement occurred in the rest 4 donor sites (3, 4, 5 and 6) with relatively longer distance to APE. To further validate sequence replacement, we performed high-throughput- sequencing of genomic DNA of F7869-transformed E . coli and obtained about 6,000,000 clean reads (~ 150 bp) in each replicate. Seamless sequence replacement was detected in these reads. As shown in Fig. 2 C, among the reads that mapped to the target region, recombinant reads, that accounted for around 1% and 0.5% (recombinant reads/total reads), were produced by the first and second donor sites from MsDFID donor template, respectively. Here, regarding a specific target site, for example, the first or second target site, total reads include those identical to the corresponding target site (wild type) or donor site sequence, respectively, and recombinant ones. The insertion and deletion (InDels) in the knock-in junctions are usually caused by NHEJ-mediated knock-in. So, to further determine the knock-in editing accuracy of this strategy, we examined the InDels in knock-in junction regions and genome-wide off-target via global screening of the high-throughput-sequencing reads. However, neither sequence-read which probably resulted from NHEJ-knock-in InDels in knock-in junctions nor unwanted off-target integration was observed (Table S3). This demonstrated that rate of NHEJ-based random integration and off-target is at a very low level, and MsDFID donor template-APE fusion structure may not promote error-prone NHEJ-mediated repair mechanism, therefore indicating high specificity of this sequence replacement system. Collectively, MsDFID donor template might function as guider and donor template, so we name it hereafter MsDFID guider and donor template (gdt). To further demonstrate its generality and editing accuracy, we employed the above-described strategy in diverse applications. We constructed other three donor vectors, CY7869-5, CY7869-9 and CY7869-21, all of whose MsDFID gdts in target dctA gene of E . coli (Fig. S2A-C). In addition, vector CY7869-6 was constructed to contain donor template also targeting dctA gene but with only one donor site with DNA fragment insertion (Fig. S2A). The sequence replacement was then detected by sequencing genotyping PCR products amplified from genomic DNA of transformed E. coli . Vectors CY7869-5, CY7869-9 and CY7869-21 can initiate expected sequence replacement in genomic target region in E. coli (Fig. S2A-C). Interestingly, no expected genotyping PCR product was amplified using the primer set of CY-2F1 and CYR1 and the genomic DNA of CY7869-6-transformed E . coli as template (Fig. S2A). This observation revealed that donor vector CY7869-6 could not prime sequence replacement as the other three vectors. Therefore, multiple DNA fragment insertion/deletion as donor sites in MsDFID template prove to be a necessary prerequisite for effective sequence replacement in this fusion strategy. We constructed another donor vector CY7869-25 to target another functional gene fadD of E. coli and then also detected expected sequence replacement in the target gene (Fig. S2D). We also constructed donor vector CY7869 in which, two MsDFID gdts targeting E . coli gene dctA and aroA , respectively, were in tandem and fused to a single APE (Fig. S3). As a result, the APE in CY7869 can mediate simultaneous seamless sequence replacement in both of the two target regions (Fig. S3). 2.3 . Identification of single clones of E. coli Top10 bearing APE-mediated sequence replacement in thyA Thymidylate synthase (ThyA) in E . coli is involved in the de novo synthesis of dTTP from dUMP. Without ThyA, the cell is unable to synthesize DNA and, therefore, will not grow in minimum growth media. Thus, thyA -null E . coli mutant can be selected (positive selection) by culture in growth medium in the absence of thymine, because de novo dTTP synthesis can proceed without the need for ThyA function when thymine is provided (Wong et al., 2005 ). Here, to further assess the efficiency of sequence replacement, we applied the selection system with ThyA as the selectable negative marker for selecting single colons of E. coli with expected sequence replacement in thyA gene as previously described (Wong et al., 2005 ). We designed fusion of APE and MsDFID gdt to target thyA (Fig. 3 A). As a result, we found that pRiceSLthyA-3 which contains gdt-APE fusion can mediate precise sequence replacement in thyA , while the negative vector, pnoSLthyA-3 with only MsDFID gdt, wasn’t able to achieve sequence replacement (Fig. 3 B-C and Fig. S4). However, the sequence replacement efficiency is relatively low (Fig. 3 D). This observation further revealed that APE, can mediate precise sequence replacement. 2.4 . Genetic requirements for gdt-guided sequence replacement in E. coli Next, we sought to determine the genetic requirements for APE-mediate target sequence replacement in E . coli . As above-mentioned, we selected E . coli strain TOP10, recA- loss-of-function ( recA1 ) mutant, to exclude the possibility that endogenous RecA-dependent and independent HDR is responsible for the sequence replacement. TOP10 contains CRISPR-Cas3 system, which belongs to Type I CRISPR-Cas system and involves two protein elements for DNA targeting and cleavage, respectively: Cascade and Cas3; E . coli Cascade is a multimeric complex of 5 different Cas proteins and responsible for processing CRISPR arrays and for binding target DNA sequences through PAM and protospacer recognition, whereas Cas3, the signature protein, is responsible for cleaving and degrading target DNA (Luo et al., 2015 ). The Cascade proteins include cas8 ( cse1 ), cas11 ( cse2 ), cas7 , cas5 ( cas5e ) and cas6e , and their open reading frames (ORFs) overlap with each other in the order of cas8 - cas11 - cas7 - cas5 - cas6e , thus forming the Cascade operon under the control of cas8 promoter which is in downstream of cas3 gene expression cassette (Fig. 4 A). E . coli CRISPR-Cas3 system has been identified to mediate a long range and unidirectional genomic DNA deletion upstream of PAM without prominent off-target activity in eukaryotic cells (Luo et al., 2015 ; Li et al.,2018; Morisaka et al., 2019 ). The removal of Cas3 from E . coli CRISPR-Cas3 system would allow Cascade to bind target DNA sequences but without subsequent degradation (Luo et al., 2015 ). So, these insights lead us to wonder whether CRISPR-Cas3 system enables the endogenous APE-mediated sequence replacement in E . coli genome in this study. We first knocked out cas3 gene from TOP10 by deleting most of its genomic sequence to create loss-of-function mutant of cas3 , TOP10Δ cas3 (Fig. S5A). Then, we transformed both TOP10Δ cas3 and wild type TOP10 strain with one of above-mentioned donor vector F7869, and checked whether sequence replacement still happened in target region by sequencing genotyping PCR product (Fig. 4 B). Our results indicated no sequence replacement was observed in the transformed TOP10Δ cas3 strain, indicating that the APE-mediated sequence replacement is dependent on the endogenous cas3 gene. We then asked whether 5 Cascade proteins of E . coli are required for the desired sequence replacement in this study. Toward this aim, we simultaneously knocked out genes cas8 and cas11 , or cas11 , cas7 and cas5 , or cas5 and cas6 in a streamlining way, to generate multiplex knockout mutants of TOP10 strain, TOP10Δ cas8cas11 , TOP10Δ cas11cas7cas5 and TOP10Δ cas5cas6e , respectively (Fig. S5B-D). Surprisingly, knockout of these cas genes can’t abrogate desired sequence replacement event in all of these TOP10 mutant strains (Fig. 4 B). Thus, among E . coli CRISPR-Cas3 system, Cas3 might be the only Cas protein required for the APE-mediated targeted sequence replacement in E . coli . Meanwhile, we used the same donor vector to transform E . coli strains BL21 and BL21 (DE3) which are derived from E . coli B strain and inherently lacks CRISPR-Cas3 system (Fig. S6). As expected, we can’t detect the desired sequence replacement in target region in transformed BL21 and BL21 (DE3) strains (Fig. 4 B), consistent with the above observation of cas3 gene being required for the sequence replacement. Therefore, these results revealed that endogenous Cas3 is the only genetic requirement for the gdt-mediated sequence replacement in E. coli . 2.5 . SpCas9 can rescue loss-of-function of E. coli Cas3 for seamless sequence replacement in E. coli As Cas9 and Cas3 share functional conservatism as shown in cleaving and degrading target DNA when engineered in genome editing (Luo et al., 2015 ; Morisaka et al., 2019 ; Csörgő et al., 2020 ), we sought to test whether Cas9 protein functionally mimic Cas3 for the seamless sequence replacement. We used vector pCas9 with functional cas9 gene ( Spcas9 ) of Streptococcus pyogenes Type II CRISPR-Cas system (Fig. 5 A) and donor vector F7869 (Fig. 2 A) to double-transform the above-used E. coli strain TOP10Δ cas3 , loss-of-function mutant of Cas3. The genotyping PCR result demonstrated that SpCas9 protein can also be programmed to achieve precise sequence replacement in the target region of E. coli as Cas3 (Fig. 5 B) and thus the mechanism of genome editing is conservative between E. coli Cas3 and SpCas9. 2.6 . The sequence replacement via gdt is probably guided by RNA For all of the above donor vectors, sgdRNA, the fused structure of MsDFID gdt and APE, can be expressed into single RNA due to the activation by lacZ promoter in E. coli . So, one important question to be settled in this context is the following: the genome edition in this study is guided by RNA or DNA directly, or both? So, we modified the above-used vectors F7869 by deleting the lacZ promoter, thus generating vectors F7869M with no virtual promoter to regulate the expression of the sgdRNA in E . coli (Fig. 6 A). After transformation of E . coli using F7869M, we detected no sgdRNA expression compared with that by F7869 (Fig. 6 B), and consequently identified no sequence replacement (Fig. 6 C). This reveals preliminarily that the sequence replacement strategy via fused gdt might mainly depend on RNA, but not DNA- guided, mechanism. 3. Discussion Here we demonstrate that the simple tool by coupling MsDFID gdt and a newly identified gdt scaffold, APE, can realize precise sequence replacement. This strategy reveals feasibility of really PAMless and off-target-free genome editing. It promises to be particularly useful for installing substitutions, insertions and deletions, especially for accurate modification of genes into their alleles harboring allele-specific point mutations or seamless insertion of short regulatory elements to fine-tune gene expression. This will exhibit especial potential for broad applicability for therapy and basic research provided its efficiency would be further improved. 3.1 . The MsDFID gdt-mediated sequence replacement in this study might well represent one solution towards PAM-independence and minimizing the risk of off-target due to its high reliability DNA sequence replacement relies on delivery of a donor repair template (DRT) into the target cell for HDR of double-stranded DNA breaks (DSBs) which, in general, are repaired through either NHEJ or HDR. NHEJ is not precise and often causes random and therefore nonspecific InDels or other mutations at the junctions between target site and its flanking sequence (Lu et al., 2016). On the contrary, HDR is precise and can hence be used to achieve precise and sequence replacement for various kinds of gene modification. However, NHEJ is the predominant pathway, while HDR is relatively rare. In this study, all the targeted DNA insertion/deletion events were seamless and in the desired direction. On the other hand, high-throughput-sequencing data analysis pointed to no obvious genome-wide off-target in generating sequence replacement. This kind of precise and exclusive sequence replacement in all of target regions is in good correspondence with the known characteristics of HDR mechanism. These results implicate a dual role for MsDFID template as both guide RNA and donor template, therefore representing a new HDR mechanism for genome editing. Zuccaro et al ( 2020 ) recently reported that, in editing human embryos using CRISPR-Cas9 system, about half of Cas9-induced double-strand break is microhomology-mediated end joining, and the most common repair outcome of the breaks remain unrepaired. This led to an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, both on-target and off-target cleavage of Cas9 results in frequent chromosome loss and hemizygous InDels because of cleavage of both alleles. These findings indicate that employing those widely-used gene edition tools would pose a substantial risk of off-target, while the strategy presented in this study feature reliability and might well represent one solution to this challenge. 3.2 . The strategy of MsDFID gdt-APE fusion specifies genome modification mechanism of E. coli Cas3 or Cas9 proteins to HDR mode The main function of CRISPR-Cas system in bacteria and archaea is widely considered to protect prokaryotic cells from exogenous genetic materials, such as virus or plasmid. The structural and functional landscape of E. coli Cascade complex (CRISPR-Cas3) characterized this system as RNA-guided immune surveillance system to defense viral invasion (Dutra et al., 2007 ; Morisaka et al., 2019 ; Zhao et al., 2014 ). In fact, type I-E CRISPR-Cas system which E. coli CRISPR-Cas3 belongs to is the most extensively studied subtype. However, surprisingly perhaps, Bozic et al addressed involvement of this system in regulating endogenous genes at transcriptional level other than the canonical immune response (Bozic et al., 2019 ). Thereby, relying more on its plausibility than on direct and convincing experimental proof, the notion of E . coli CRISPR-Cas3 functioning as native defense system is circumstantial and a controversial subject so far (Mulepati et al., 2013;Sinkunas et al., 2013 ). Here we provided in vivo evidence in E . coli to implicate E . coli Cas3 in targeted modification of endogenous genomic sequence in E . coli , thereby shedding new light on those seemingly contradictory reports. Taken together, the above data allow us to propose the model in which Cas3 or Cas9 activity could be readily reprogrammed by APE to achieve precise sequence replacement (Fig. 7 ). The APE could be recognized and bound by E . coli Cas3 or Cas9 protein, thus forming MsDFID gdt/Cas3 or Cas9 ribonucleoprotein. Previous related reports revealed that E . coli CRISPR-Cas3 system, but not Cas3 protein only, when being engineered to edit the genome of other organisms, is dependent on PAM and non-HDR DNA repair mode to generate large deletions (Luo et al., 2015 ; Morisaka et al., 2019 ; Dolan et al., 2019 ). Alternatively, in this study, APE specifies genome modification mechanism of Cas3 protein to efficient HDR mode without limitation of PAM (Fig. 7 ). And sgdRNA may function as a RNA donor repair template (DRT) for homology-directed DNA repair (HDR) of double-stranded DNA breaks, a transcript-templated HDR (TT-HDR). 3.3 . The multiple DNA fragment insertion/deletion as donor sites in MsDFID template is a key genetic requirement for the sequence replacement Both crossover and non-crossover events involving chromatids during meiosis cause intragenic recombination that can generate new alleles or new combinations of existing alleles (Zhang et al., 2020 ; Zhou et al., 2020 ). In fact, meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. A higher rate of both intrachromosomal recombination (presumably via cross-over event) and mutation (mechanistically uncoupled) preferentially occur in high diversity domains in chromosomes during meiosis (Yang et al., 2015 ). Heterozygosity might well be causative for this finding, thus convincingly explaining the more diverse population-wide mutation rate variations including intragenic/allelic recombination in progeny of heterozygotes than homozygotes among F2 population (Yang et al., 2015 ). The underlying mechanism might be that heterozygosity would enhance the likelihood of poor pairing and consequently proceeding to Spo11-mediated DSBs for physically exposed regions (unsynapsed chromosomes and chromatin loops (Yang et al., 2015 ). A three-stranded nucleic acid structure termed R-loops is universally formed during transcription, as the nascent RNA molecule may hybridize with the template DNA strand, forming a DNA:RNA hybrid and leaving the nontemplated DNA single-stranded (Yang et al., 2017 ). R-loops have been found to persist throughout the genomes of various species (Yang et al., 2017 ). The common chromatin features of R-loop promote ectopic HR activity, thereby participating in a number of physiological processes, such as gene expression, DNA replication, and DNA and histone modifications, and DNA damage repair and genome stability (Yang et al., 2017 ; Sun et al., 2017 ). On the other hand, R-loops can lead to DNA damage as the single-strand DNA (ssDNA) formed from RNA/DNA hybridization is susceptible to mutagenes and lesions, leading to double-strand breaks and ectopic recombination that further induces genome instability (Yang et al., 2017 ). Similarly, a sequential double-strained DNA (dsDNA) target searching and degradation process by Type I-E CRISPR Cascade of Thermobifida fusca ( TfuCascade ) feature the unwinding of entire protospacer to form a full R-loop which triggers conformational changes in Cascade, licensing Cas3 to bind (Xiao et al., 2017 ). In fact, in the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The structure provides the necessary temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The negative and positive checkpoints coordinately ensure stringent yet efficient target degradation in type I CRISPR-Cas systems (Xiao et al., 2017 ). From these knowledge, we suppose that in searching target dsDNA, the DNA fragment insertion/deletion array which features MsDFID gdt, could enhance the heterozygosity between gdt and its target strand. This would enable the formation of multiple bulges in gdt or its target strand (complementary strand) when they pairs with each other. Consequently, these bulges probably promote the priming of efficient HDR but not canonical NHEJ mode for sequence replacement by Cas3 or Cas9 proteins in E . coli . In other words, these bulges might well act as key rate-limiting factors to improve HDR efficiency and in turn HDR-mediated sequence replacement. Notably, among the multiple DNA insertion/deletions (donor sites) in MsDFID gdt, the closer the one is to APE, the higher sequence replacement rate in the respective target site. So, for desired efficient sequence placement, DNA fragment insertion/deletion array need to be designed to form MsDFID gdt, but the donor site of interest should be arranged as the nearest to APE. This will achieve the sequence replacement only in its respective target site with the highest likelihood, thereby making the selection process as easy as possible. 3.4 . Functional conservation between E. coli Cas3 and SpCas9 The enormous diversity of viruses and their complex patterns of coevolution with defense systems suggests that more types of defense systems with diverse mechanisms can be expected to exist than are currently known (Pausch et al., 2020 ; Gao et al., 2020 ). In fact, in the context of an incessant arms race with mobile genetic elements, evolution of CRISPR-Cas systems has resulted in extreme diversification of both Cas protein sequences and architecture of the CRISPR-Cas loci (Shmakov et al., 2017 ). Given such an exciting forefront, considerable effort focused on exploring more CRISPR-Cas or CRISPR-Cas-like systems for providing more powerful addition to genome editing toolbox; whereas we devoted our efforts to dissecting and harnessing the cross-compatibility of different CRISPR-Cas systems. Obviously, the engineering of fusing MsDFID gdt to APE in this study is a kind of simplified strategy for genome editing. Although E . coli Cas3 and SpCas9 proteins are derived from evolutionally divergent eubacteria, both of them can recognize the newly discovered gdt scaffold, APE. This revealed a kind of conservation in functional mechanism between the two Cas proteins which represent class 1 (type I) and class 2 systems (type II), respectively. Importantly, the knowledge will facilitate heterologous reconstitution of other well-characterized Cas proteins and MsDFID gdt to establish HDR as only/main genome edition mechanism for reprogrammable sequence replacement. 4. Materials and methodology 4.1 . Used E. coli strains The used E . coli strains are listed in Supplementary Table S1 . 4.2 . E. coli growth conditions For strain and plasmid construction, E . coli strains were cultured in Luria-Bertani (LB) medium except for special requirement. For fermentation, strains were cultured in 50 mL M9 medium containing 2 g/L Amicase (Sigma, St. Louis, MO, USA), 0.2 g/L l-arabinose, and 1% (v/v) glycerol; 3% glycerol was used for PHB production. To maintain the plasmids, final concentrations of 100 µg/mL ampicillin, 50 µg/mL spectinomycin, and 25 µg/mL chloromycetin were added to the corresponding cultures. 4.3 . Construction of sgdRNA expression vectors (donor vectors) Fused gdRNA and scaffold APE, sgdRNAs, were synthesized and cloned into pUC57 vector, thereby yielding donor plasmid F7869, pRiceSLthyA-3 and other expression vectors. For F7869M, fused gdRNA and scaffold APE was cloned into pUC57m (modified pUC57 vector) without actual promoter near the multi-cloning site (MCS). The sequences of target region and donor sites regarding F7869/F7869M are listed in Table S2. pCas9 vector with a stable expression system of functional SpCas9 in E . coli , was purchased from Addgene, a non-profit plasmid repository. 4.4 . Application of ThyA selection system for identifying single clones of E. coli bearing efficient and seamless DNA sequence replacement in thyA Two E . coli strains, TOP10 and TOP10Δ cas3 , loss-of-function mutant of cas3 of TOP10, were used in this experiment. Cells were grown in a modified M9 minimal medium as previously described, containing 48 mM Na 2 HPO 4 7H 2 O, 22 mM KH 2 PO 4 , 8.6 mM NaCl and 18.7 mM NH 4 Cl, 1 mM MgSO 4 , 0.5 mM CaCl 2 , 0.4% (w/v) glucose, 0.6 mM leucine, valine and isoleucine, 30 nM vitamin B1, 1% (v/v) Luria-Bertani (LB) broth and 1×MM1 containing 1 nM each of ZnSO 4 , CuSO 4 , Na 2 MoO 4 , CoCl 2 , MnSO 4 , CrCl 3 and NiCl 2 (Wong et al., 2005 ). Specific growth selection medium was supplemented with either or combination of 50 mg/ml ampicillin, 12.5 mg/ml chloramphenicol, 0.79 mM thymine (Sigma) and 10 mg/ml (for plasmids) trimethoprim (Sigma). For growth on agar plates, 2.5% (w/v) noble agar (Sigma) was included in the appropriate medium. All chemicals were of analytical grade. Appropriate E . coli cells were prepared for electroporation as described previously (Wong et al., 2005 ). In brief, 50 ml of ‘competent’ cells were mixed with 2–8 mg of plasmids in a pre-cooled 0.1 cm Gene Pulser cuvette (Bio-Rad Laboatories) and electroporated using the Bio-Rad Gene Pulser with conditions set at 1.8 kV, 25 mF and 200 ohms. Following electroporation, the cells were grown in 1 ml of LB broth at 37°C for 1-1.5 h, collected by centrifugation and plated at various dilutions onto M9 minimal medium agar plates with appropriate supplements for selective growth, and incubated at 37°C for 48–72 h. 4.5 . Identification of sequence replacement in target region Genomic DNA from cell pellets of single clones of transformed E . coli was extracted using Ezup Column Bacteria Genomic DNA Purification Kit according to the manufacturer’s protocol (Sangon Biotech Co. Ltd., Shanghai, China). Genomic PCR was performed using Green Taq Mix containing High-Fidelity Taq DNA polymerase DNA、dNTP and optimized buffer (Q5) according to the manufacturer’s protocol (Vazyme Co. Ltd., Nanjing, China). To genotype the types of sequence replacement in target regions, the PCR products were purified and then subjected to direct DNA Sanger-sequencing service, or ligated into pUC18-T T-vector for transformation of E . coli and then Sanger-sequencing plasmid. All of used primers are listed in Supplementary Table S4. 4.6 . High-throughput sequencing of genomic DNA and analysis 4.6.1. Total DNA extraction and quality control Total genome DNA from transformed E . coli by donor vectors was extracted using CTAB/SDS method. The integrity of DNA was measured by using 1% gel electrophoresis. DNA concentration and purity were measured by the NanoDrop 200 Spectrophotometer (Thermo Fisher, USA), and OD260/280 was 1.8-2.0. DNA concentration was measured by Qubit 3.0 Fluorometer (Life Technologies, USA). 4.6.2. Library preparation DNA sequencing libraries were generated using the KAPA-HyperPlus Kit according to the manufacturer’s protocol (The extracted DNA was broken into fragments, then terminal repair, 3' terminal addition, ligation, and addition of primers, perform size selection, PCR amplification and purification, and quality inspection of every library.). Sequencing was performed by the Bioacme Biotechnology Co., Ltd. (Wuhan, China). The resulting libraries were size-checked by using an Agilent 2100 Bioanalyzer system (Agilent). Finally, the library preparations were sequenced on an Illumina NovaSeq platform and 150 bp paired-end reads were generated. 4.6.3. Data analysis Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from E . coli genome K-12. Clean reads were aligned to the reference genome using short read mapping software BWA. As a result, approximately 99.99% of the reference genome was covered with uniquely mapping, non-duplicate reads, to an average coverage depth of around 200X. Reads were mapped onto the reference genome and single nucleotide polymorphisms (SNPs) or InDels were detected by BCFtools software. The SVs (Structural Variations) were identified using DELLY2. The copy number variations of every sample were using CNVnator software. The detected variant sites were annotated using ANNOVAR. 4.7. Knockout of E. coli Cas proteins The knockout of cas3 , cas8-cas11 , cas11-cas7-cas5 and cas5-cas6e in E . coli strain TOP10 was conducted by using coupled CRISPR/Cas9 technology and λ Red homologous recombination system as previously described (Jiang et al., 2015 ). The resulting loss-of-function mutant strains, TOP10Δ cas3 , TOP10Δ cas5cas6e , TOP10Δ cas8cas11 and TOP10Δ cas11cas7cas5 were verified by PCR and agarose gel electrophoresis. 4.8. RNA extraction and semi-quantitative RT-PCR Total RNA was extracted from plant tissues using TRIzol reagent (Life Technologies) and treated with RNase-free DNase I (Invitrogen). Two micrograms of RNA were reverse transcribed using oligo (dT) primer and AMV reverse transcriptase (Promega). Quantitative PCR experiments were performed. Each qRT-PCR assay was replicated at least three times with three independent RNA preparations. The E . coli 16S rRNA was used as internal control. Primers used are listed in Supplementary Table S4. 4.9. Statistics An unpaired two-sided Student’s t -test was implemented for group comparison as indicated in the Fig. legends. Declarations CRediT authorship contribution statement Rui Li : Writing-review & editing, Writing-original draft, Data curation, Formal analysis, Resources. Zeyu Qi : Writing-original draft, Formal analysis, Data curation, Conceptualization, Methodology. Yueyang Wanghuang : Data curation, Formal analysis, Resources. Yonghan Xu : Writing-review & editing, Writing-original draft, Methodology, Funding acquisition, Investigation, Data curation, Conceptualization, Project administration, Supervision. Dechuan Wu : Writing-original draft, Methodology, Data curation. Wei Tang . Software, Methodology, Formal analysis, Data curation. Xiang Chen : Data curation, Investigation, Methodology. Xiaoyu Hu : Conceptualization, Data curation, Methodology, Resources. Guoxing Zou : Investigation, Resources, Writing- review & editing. Jiahao Hua : Data curation, Formal analysis, Methodology, Resources. Zhangying Feng : Data curation, Formal analysis, Methodology, Resources. Xuelan Zong : Data curation, Formal analysis, Methodology, Resources. Qiutao Hu : Data curation, Formal analysis, Methodology, Resources. Jingcai Li : Conceptualization, Funding acquisition, Project administration, Resources, Supervision. Xinchun Lin : Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing-original draft, Writing-review & editing. Xiaobo Wang : Conceptualization, Project administration, Resources, Supervision, Writing-review & editing. Chuanxi Ma : Conceptualization, Funding acquisition, Project administration, Resources, Supervision. Ethics declarations Ethical approval and consent to participate Not applicable. This article does not include any studies conducted by the authors involving human participants or animals. All research and analyses presented are based on experimental methods and data that do not include human or animal subjects. This disclaimer ensures compliance with ethical standards and regulations regarding research involving human and animal participants. Consent for publication Not applicable. Competing interests The authors declare no competing interests. Declaration of competing interest The authors declare no competing interests. The views and opinions expressed in this article are purely those of the authors and do not necessarily reflect the official policy or position of author's employer or any Novartis officers. Data availability Data will be made available on request. Acknowledgements We thank Prof. Yang Sheng for technical support help in developing ThyA selection system to identify single clones of E . coli bearing seamless DNA sequence replacement in thyA, Prof. Yongshen Liu for support in experiment design technical support and Prof. Songhu Wang for critical reading of the manuscript and discussion. This work is supported by University Collaborative Creation Project of Anhui Province to C.X.M. (grant no. GXXT-2019-033). Y.H.X. was supported by Anhui Provincial Nature Science Foundation (No. 1808085MC87), a startup fund (No. 2018008) from Anhui Agricultural University, and Industry Alliance Project of Dabie Mountain Experimental Station of Anhui Agricultural University (No. 202408). W. T. was supported National Natural Science Foundation of China (No. 31700266). 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Zuccaro MV, Xu J, Mitchell C, Marin D, Zimmerman R, Rana B, Weinstein E, King RT, Palmerola KL, Smith ME. Allele-specific chromosome removal after Cas9 cleavage in human embryos. Cell. 2020;183:1650–64. Additional Declarations No competing interests reported. Supplementary Files Supplementaldata202509.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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13:45:09","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90760,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/e2f73f397231528600417fca.png"},{"id":94200474,"identity":"0da3fdd1-9727-4e6a-91a9-f79489f9dbe7","added_by":"auto","created_at":"2025-10-23 13:53:09","extension":"xml","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":136801,"visible":true,"origin":"","legend":"","description":"","filename":"434f35d6f1c94cc09a04020a68072d071structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/df71aac6219fd62b27f5346c.xml"},{"id":94200112,"identity":"ce384d1c-68c0-4c02-b2d8-1b71e8d00e59","added_by":"auto","created_at":"2025-10-23 13:45:09","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":155872,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/e3fae27a53e14eb1152628c3.html"},{"id":94200099,"identity":"b8dd9bb7-a120-41a9-8824-6789c870aff5","added_by":"auto","created_at":"2025-10-23 13:45:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":12121476,"visible":true,"origin":"","legend":"\u003cp\u003eStructure of APE. (A) Location of the APE in the promoter region of rice gene\u003cem\u003e IPA\u003c/em\u003e. The numbers under the genome sequence denote the location of indicated genes and APE8.1. (B) Structural detail of overall organization of the APE from the promoter region of rice gene \u003cem\u003eIdeal Plant Architecture1 \u003c/em\u003e(\u003cem\u003eIPA1\u003c/em\u003e). The direct and reverse repeat units are indicated in green and pink. The variant base in the repeat units is highlighted in red.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/40c5ec3150e09eb2753ca4b1.jpeg"},{"id":94201610,"identity":"75519a5d-e4c4-410e-9578-98e0476fc2d2","added_by":"auto","created_at":"2025-10-23 14:01:08","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":17914342,"visible":true,"origin":"","legend":"\u003cp\u003eMsDFID gdt-APE-fusion mediates efficient sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e gene \u003cem\u003earoA\u003c/em\u003e. (A) Sequence replacement strategy targeting \u003cem\u003earoA\u003c/em\u003e by donor vector F7869. Left, compared with its target region, MsDFID gdt (solid boxes) in vector F7869 possesses 6 donor sites（1-6）including 5 sites (1-5) with DNA fragment insertion (red loops) and a site with deletion of 12 bp DNA fragment (red dashed loop) (the sixth). Each homologous arm is 60 bp in length. The loops are supposed to form when putative homologous pairing happens between MsDFID gdt (donor template) and its genomic target region. Schematics also indicate the location of genotyping primers for genotyping PCR. Right, a schematic summary of precise sequence replacement in target gene \u003cem\u003earoA\u003c/em\u003e. The two types of flanking PCR amplicons correspond to two types of DNA sequence replacement events in target site 1 and 2 in gene \u003cem\u003earoA\u003c/em\u003e, respectively, as shown in the bottom of (B). (B) Identification of precise sequence replacement in target gene \u003cem\u003earoA\u003c/em\u003e. Top left, agarose gel electrophoresis of flanking PCR (genotyping PCR) product amplified using genomic DNA of F7869-transformated \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli \u003c/em\u003eas template and flanking primer set of FF2 and FR4 which are specific for flanking genomic sequence of target region in \u003cem\u003earoA\u003c/em\u003e gene as shown in (A). Lanes 1-8, positive genotyping PCR result with genomic DNA of 8 single clones of F7869-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e as PCR template. Lane 9: negative control. M: DNA ladder marker. For the panel, n=3 replicates for PCR reactions were performed. Top right, Sanger-sequencing chromatogram of total flanking PCR product as shown in Top left. In the site noted by red arrowhead, well-marked double peak of chromatogram appeared, an evidence of being heterozygotes of wild-type and replacement-edited sequence. This meant that the insertional DNA fragment in donor site 1 of MsDFID gdt as shown in top left of (A) had been knocked into the desired genomic target site (indicated by arrowhead). The inserted DNA fragment of 9 bp from donor site 1 was colored in red. For all panels, all genotyping PCR reactions were performed in at least 3 replicates. Bottom, Sanger-sequencing chromatogram of three kinds of flanking PCR product which defined the genotypes of the wild type, type 1 and type 2 in the target region. (C) \u0026nbsp;Summary of seamless sequence replacement frequency in target region in the genome of F7869-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli. \u003c/em\u003eThe percentages in the \u003ca href=\"http://www.baidu.com/link?url=0QTN-T9vBeyt29hKafFaePAcm8pPla1Cv2TDaX-3aXFPwLXMbzY22SO7Kc0XxP3DQJaBWztvyiR5JRMjOE30gleH4qUUyqXUUhMQqKZIetG\" target=\"_blank\"\u003eparentheses\u003c/a\u003e behind the read No. indicated the proportion of recombinant reads in the total reads mapped to the indicated target site. Here, total reads include those identical to the corresponding target site (wild type) or donor site sequence along with homologous arms, respectively, and recombinant reads.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/fc974da46e43ef20f5d94de3.jpeg"},{"id":94202508,"identity":"7add4e5a-40db-43e2-b2ca-b147352d4a52","added_by":"auto","created_at":"2025-10-23 14:09:20","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3112060,"visible":true,"origin":"","legend":"\u003cp\u003ePositive selection and confirmation of single clones bearing target sequence replacement in \u003cem\u003ethyA \u003c/em\u003egene of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Top10\u003cem\u003e.\u003c/em\u003e (A) Construct of MsDFID donor template in pRiceSLthyA-3 vector targeting \u003cem\u003ethyA\u003c/em\u003e gene in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. The donor sites in MsDFID template, including DNA fragment insertion and deletion sites, were indicated by blue and red, respectively. ThyA-3outF3 and thyA-3outR3 are the primers used for genotyping PCR flanking PCR. (B) Screening of positive single clones via\u003cem\u003e \u003c/em\u003eThyA positive selection of Top10 strains\u003cem\u003e \u003c/em\u003etransformed by vector pRiceSLthyA-3 and pnoSLthyA-3, respectively. Please note that no single clone transformed by control vector pnoSLthyA-3 had grown in the selection medium. 10\u003csup\u003en \u003c/sup\u003eis the dilute multiple (dilution factor) of transformants on the indicated selection medium or control medium. (C) Sequence comparison of target region in \u003cem\u003ethy\u003c/em\u003eA gene between the wild type and pRiceSLthyA-3 transformed Top10 strain. M1-M3, positive transformants with different sequence replacement events. Please refer to Fig. S4 for the sequencing chromatogram of genotyping PCR product confirming sequence replacement. (D) Sequence replacement efficiency of \u003cem\u003ethyA \u003c/em\u003egene in Top10 strain transformed by pRiceSLthyA-3 or pnoSLthyA-3 vector. The rate (means ± SD) of single clones carrying the desired sequence replacement to total clones was calculated as the editing efficiency from three independent experiments (n=3). \u003cem\u003eP\u003c/em\u003e-value was calculated using a two-sided \u003cem\u003et\u003c/em\u003e-test. ***\u003cem\u003eP\u003c/em\u003e\u0026lt;0.001: extremely significant difference.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/d1399516edc832753fb032b2.jpeg"},{"id":94200094,"identity":"69750ca8-ceca-4488-84e7-3dea36db3a03","added_by":"auto","created_at":"2025-10-23 13:45:08","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":183090,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of genetic association with the availability of APE-mediated sequence replacement in different \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli \u003c/em\u003estrains.\u003cem\u003e \u003c/em\u003e(A) Schematic genomic organization of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 locus. P\u003csub\u003e\u003cem\u003ecas3\u003c/em\u003e\u003c/sub\u003e and P\u003csub\u003e\u003cem\u003ecas8\u003c/em\u003e\u003c/sub\u003e represent promoters of \u003cem\u003ecas3\u003c/em\u003e and Cascade operon, respectively. (B) Identification of sequence replacement in the various transformed\u003cem\u003e E\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains. The used plasmid F7869 for transforming \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains contained APE. The wild type TOP10 strain was also transformed with the same plasmid to be used as positive control. The genetic background of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains used in this experiment is indicated. “–” is negative control.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/0e37de596bd5031b724e415f.jpeg"},{"id":94200470,"identity":"c7919c33-8428-447b-bf6d-dc9695eaf575","added_by":"auto","created_at":"2025-10-23 13:53:08","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":500332,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional complementation of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003eCas3 by SpCas9. (A) A schematic map of \u003cem\u003eCas9\u003c/em\u003eexpression vector. (B) (Top) MsDFID gdt (solid boxes) in donor vector F7869 and the location of primers for genotyping PCR as follows. (Bottom): Investigation of sequence replacement in the TOP10Δ\u003cem\u003ecas3\u003c/em\u003e strain double-transformed by SpCas9 expression vector pCas9 and donor vector F7869. F7869-transformed TOP10 which harbors endogenous CRISPR-Cas3 system, was used as positive control.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/5c14369125ff907be4c549df.jpeg"},{"id":94200097,"identity":"f35535a9-58be-4458-be96-0b2089bea1e7","added_by":"auto","created_at":"2025-10-23 13:45:08","extension":"jpeg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":915167,"visible":true,"origin":"","legend":"\u003cp\u003eConfirmation of sequence replacement guided by MsDFID gdt. (A) Schematic description of construction of donor vectors F7869M. For the multi-cloning site (MCS) of pUC57-Mini plasmid, there is no any adjacent promoter for activating the transcription of MsDFID gdt. The synthesized MsDFID gdt-APE fusion was ligated into the MCS of pUC57-Mini, thereby generating plasmid F7869M. (\u003cstrong\u003eB\u003c/strong\u003e) Expression of sgdRNA in F7869M-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e TOP10. \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e 16S rRNA was used as internal control. The used semi-quantification primer sets here are FM(F7869)-F2 and FM(F7869)-R1, 16SrSemi-RTF1 and 16SrSemi-RTR1, for gdt and 16S rRNA (\u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e), respectively. (\u003cstrong\u003eC\u003c/strong\u003e) No sequence replacement in the target region in F7869M-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. The genotyping primers were described in Fig. 5. In both (\u003cstrong\u003eB\u003c/strong\u003e) and (\u003cstrong\u003eC\u003c/strong\u003e),“–” is negative control.\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/6b071ab0cb1e62de842d504d.jpeg"},{"id":94200471,"identity":"f74e482b-cb40-48f8-8f8c-67fa4c127d05","added_by":"auto","created_at":"2025-10-23 13:53:08","extension":"jpeg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":383084,"visible":true,"origin":"","legend":"\u003cp\u003eProposed model for APE-mediated precise sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. Here, in the MsDFID gdt/Cas3 or MsDFID gdt/Cas9 ribonucleoprotein, APE might act as scaffold of MsDFID gdt reprograms \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cas3 (Left) or Cas9 (Right) for precise and almost off-target-free sequence replacement without requirement of PAM.\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/d854ba7a4298214fcec80fe2.jpeg"},{"id":99315458,"identity":"8ebf4c25-db80-4fbc-b30c-1141e72fbeda","added_by":"auto","created_at":"2025-12-31 16:26:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":36372045,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/a70b16a9-7151-432c-ae70-5ba9375162b3.pdf"},{"id":94200115,"identity":"ebca53b0-5e13-410d-8880-f78fccec4806","added_by":"auto","created_at":"2025-10-23 13:45:10","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":28126720,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaldata202509.doc","url":"https://assets-eu.researchsquare.com/files/rs-7640593/v1/c83a0d647f53ebfc62ac8cd0.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eFor CRISPR-Cas9 systems, it is possible to achieve precise integration of new DNA following Cas9 cleavage either through homologous recombination (HR) (Yu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Jasin et al., 2013) or error-prone non-homologous end joining (NHEJ) (Schmid-Burgk et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Suzuki et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, requirement of the recognition of protospacer adjacent motif (PAM) limits target site recognition to a subset of sequences in DNA manipulation by CRISPR-Cas enzymes. To overcome this constraint, Walton et al (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) engineered specific variant of \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Cas9 (SpCas9) that could target almost all PAMs, thus eliminating NGG PAM requirement. Strecker et al reported Tn7-like transposases subunit(s) from cyanobacteria can be reprogrammed through association with type V-K CRISPR effector (Cas12k) to insert DNA into \u003cem\u003eE. coli\u003c/em\u003e genome (Strecker et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The reconstitution strategy can achieve unidirectional insertion of DNA segment of up to 2.5 kb into unique sites downstream of protospacer with frequencies of up to 80% but without positive selection. Nevertheless, this does not belong to means of precise sequence replacement. Yu et al (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) described a strategy which realized gene knock-in rate of up to 65/40% for 0.7/2.5 kilobase inserts, at various genomic loci in human cancer and stem cells, respectively, via using Cas9 and 5\u0026prime;-modified double-stranded DNA as donor template. Similarly, Lu et al (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) employed this strategy to insert sequences of up to 2.049 kilobase pairs into rice genome at efficiency of 25%. However, the two cases of sequence replacement relied on specificly designed schemes and sophisticated operations with the limitation of PAM and risk of off-target. Prime editors are CRISPR-Cas9 nickase (H840A)-reverse transcriptase accompanied with prime editing guide RNAs and can generate base conversions, and small insertions and deletion in plant and animal cells without donor DNA or double-strand breaks (Anzalone et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lin et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Further, Sun et al (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) developed a third-generation PrimeRoot editors which employ optimized prime editing guide RNA designs, an enhanced plant prime editor and superior recombinases to enable precise large DNA insertions of up to 11.1 kilobases into plant genome. But, all of these strategies face issues of PAM requirement and off-target. So, precise, PAMless and off-target-free sequence replacement still remains practically challenging. Here, via fusing donor sequence targeting genome site and a rice-derived AT-rich pincer-like elements (APE), we realized the precise sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. In this system, APE establishes a dual role of the donor sequence as both guider and donor template (gdt). The knock-out experiment of Cas genes \u003cem\u003ecas3\u003c/em\u003e, or \u003cem\u003ecas8\u003c/em\u003e and \u003cem\u003ecas11\u003c/em\u003e, or \u003cem\u003ecas11\u003c/em\u003e, \u003cem\u003ecas7\u003c/em\u003e and \u003cem\u003ecas5\u003c/em\u003e, or \u003cem\u003ecas5\u003c/em\u003e and \u003cem\u003ecas6\u003c/em\u003e, in a streamlining way, confirmed that, among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 system, Cas3 might be the only Cas protein required for the APE-mediated targeted sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. Meanwhile, gdt-APE fusion can also directed Cas9 to achieve precise sequence replacement in \u003cem\u003ecas3\u003c/em\u003e-knockout \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strain. This simplifies the tool from three components (an enzyme, guide RNA and donor DNA/RNA) to just a cutting enzyme, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cas3 or Cas9, and gdt. This simplicity in combination with minimal off-target and no PAM requirement will probably make the tool particularly advantageous for easier vector-based delivery into cells, a wider range of targetable genomic sequences and more purposes of genome editing.\u003c/p\u003e\u003cp\u003eWe thus conclude that endogenous Cas3 can be programmed to achieve precise sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.1\u003c/em\u003e. \u003cem\u003eStructure of AT-rich pincer-like elements (APE) from rice\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eWe found that the promoter region of rice gene \u003cem\u003eIdeal Plant Architecture1\u003c/em\u003e (\u003cem\u003eIPA1\u003c/em\u003e) harbors a kind of unique structure, AT-rich pincer-like elements (hereafter named APE) (526-bp in length, -1190 to -1715 bp from start code ATG) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). The APE consists of 8 direct and 9 reverse repeats of 13 bp which are interspaced by short DNA fragments of 16\u0026ndash;19 bp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In fact, both the 8 direct repeats and spacers are basically complementary to their respective reverse repeats and spacers, respectively, in the APE (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). There are only 9 single nucleotide polymorphisms (SNPs) across all of these 17 repeats (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). So, this sequence, with A/T bases as high as 74%, is predicted to be able to form a symmetric hairpin structure with several large or small loops in two stems (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In fact, APEs were predicated to form various folding patterns at RNA secondary structure level (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e2.2\u003c/em\u003e. \u003cem\u003eFusion of MsDFID guide and donor template (gdt) and APE primes precise sequence replacement in E. coli\u003c/em\u003e\u003c/p\u003e\u003cp\u003eAlthough no obvious target of APE spacers was found, we still wonder whether APEs can mediate genome editing. To test the hypothesis, we synthesized a DNA sequence which is designed as donor template against target gene \u003cem\u003earoA\u003c/em\u003e (3-phosphoshikimate 1-carboxyvinyltransferase, NP_309018.1), a functional gene of \u003cem\u003eE. coli\u003c/em\u003e. Compared with its target region of an around 350 bp in \u003cem\u003earoA\u003c/em\u003e, the synthesized sequence contains multi-sites (6 sites) with DNA fragment insertion/deletion (MsDFID) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table S2). These 6 sites act as donor sites, among which 5 and 1 sites harbor DNA fragment insertion (9 bp, 15 bp, 21 bp, 30 bp and 40 bp, in length, respectively) and 12 bp deletion, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Table S2). Then the MsDFID sequence was fused with APE, thus forming MsDFID donor template-APE fusion which was ligated into the downstream of \u003cem\u003elacZ\u003c/em\u003e promoter in modified T-vector pUC57 (hereafter named pUC57m) to yield vector F7869 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). So, F7869, which possesses MsDFID donor template, might be a donor vector.\u003c/p\u003e\u003cp\u003eMost reviews on genetic recombination support the statement that, in bacteria, all of homology-directed DNA repair (HDR) pathways are primarily dependent on RecA protein (Dutra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). So, to rule out the possibility of endogenous RecA-dependent HDR, we selected \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strain including TOP10 or T1 which bears loss-of-function \u003cem\u003erecA\u003c/em\u003e gene for investigating sequence replacement event thereafter in this study. Meanwhile, RecA-independent recombination, which relies upon the knockout of exonucleases including ExoI, ExoVII, ExoX, and RecJ, was also ruled out, because these exonucleases is efficient in these two strains (Dutra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo check whether MsDFID donor template-APE fusion mediates sequence replacement in the target region in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e genome, we isolated the genomic DNA of F7869-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. Desired sequence replacement (DNA fragment insertion or deletion) in target region is expected to produce larger or smaller amplicons. We then performed genotyping PCR with genomic DNA of transformed \u003cem\u003eE. coli\u003c/em\u003e. Insertion or deletion frequency was also quantitated for all the 6 target sites. To exclude the contamination of plasmid DNA as PCR template, the used primer set, FF2 and FR4, were designed to be specific for the genomic sequences of \u003cem\u003earoA\u003c/em\u003e flanking the homologous arms, thus ensuring genotyping PCR (namely flanking PCR) product to be \u003cem\u003eE. coli\u003c/em\u003e genome-specific but not for the donor vector (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The resulting genotyping PCR product was Sanger-sequenced and the chromatogram results showed that the clear double peaks appeared just corresponding to the targeting position of No.1 donor site with a DNA insertion in the MsDFID donor template of vector F7869 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The double peaks demonstrated that the PCR amplicons were heterozygous and consists of wild-type and edited sequences in genomic target region, therefore indicating that some genomic DNAs had been successfully edited (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Next, the total genotyping PCR amplicons were ligated into T-vector and the resulting plasmids were used to transform \u003cem\u003eE. coli\u003c/em\u003e. The 40 single \u003cem\u003eE. coli\u003c/em\u003e clones were collected to extract the recombination plasmids for Sanger-sequencing inserted PCR amplicons. Sequencing result identified two types of PCR amplicons which defined two independent sequence replacement events between No.1 and No. 2 donor sites in MsDFID donor template and their respective target sites in \u003cem\u003earoA\u003c/em\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Sequence replacement rates were measured up to 22.5% and 2.5% for the two cases, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Clearly, the replacement efficiency in No.1 donor site is much higher than that in No.2, and thus inversely proportional to the distance of the respective donor site of MsDFID donor template to APE in donor vector. Meanwhile, possibly due to limited number of selected \u003cem\u003eE. coli\u003c/em\u003e single clones for sequencing inserted PCR amplicons, we failed to identify the sequence replacement occurred in the rest 4 donor sites (3, 4, 5 and 6) with relatively longer distance to APE.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further validate sequence replacement, we performed high-throughput- sequencing of genomic DNA of F7869-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e and obtained about 6,000,000 clean reads (~\u0026thinsp;150 bp) in each replicate. Seamless sequence replacement was detected in these reads. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, among the reads that mapped to the target region, recombinant reads, that accounted for around 1% and 0.5% (recombinant reads/total reads), were produced by the first and second donor sites from MsDFID donor template, respectively. Here, regarding a specific target site, for example, the first or second target site, total reads include those identical to the corresponding target site (wild type) or donor site sequence, respectively, and recombinant ones.\u003c/p\u003e\u003cp\u003eThe insertion and deletion (InDels) in the knock-in junctions are usually caused by NHEJ-mediated knock-in. So, to further determine the knock-in editing accuracy of this strategy, we examined the InDels in knock-in junction regions and genome-wide off-target via global screening of the high-throughput-sequencing reads. However, neither sequence-read which probably resulted from NHEJ-knock-in InDels in knock-in junctions nor unwanted off-target integration was observed (Table S3). This demonstrated that rate of NHEJ-based random integration and off-target is at a very low level, and MsDFID donor template-APE fusion structure may not promote error-prone NHEJ-mediated repair mechanism, therefore indicating high specificity of this sequence replacement system.\u003c/p\u003e\u003cp\u003eCollectively, MsDFID donor template might function as guider and donor template, so we name it hereafter MsDFID guider and donor template (gdt).\u003c/p\u003e\u003cp\u003eTo further demonstrate its generality and editing accuracy, we employed the above-described strategy in diverse applications. We constructed other three donor vectors, CY7869-5, CY7869-9 and CY7869-21, all of whose MsDFID gdts in target \u003cem\u003edctA\u003c/em\u003e gene of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e (Fig. S2A-C). In addition, vector CY7869-6 was constructed to contain donor template also targeting \u003cem\u003edctA\u003c/em\u003e gene but with only one donor site with DNA fragment insertion (Fig. S2A). The sequence replacement was then detected by sequencing genotyping PCR products amplified from genomic DNA of transformed \u003cem\u003eE. coli\u003c/em\u003e. Vectors CY7869-5, CY7869-9 and CY7869-21 can initiate expected sequence replacement in genomic target region in \u003cem\u003eE. coli\u003c/em\u003e (Fig. S2A-C). Interestingly, no expected genotyping PCR product was amplified using the primer set of CY-2F1 and CYR1 and the genomic DNA of CY7869-6-transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e as template (Fig. S2A). This observation revealed that donor vector CY7869-6 could not prime sequence replacement as the other three vectors. Therefore, multiple DNA fragment insertion/deletion as donor sites in MsDFID template prove to be a necessary prerequisite for effective sequence replacement in this fusion strategy.\u003c/p\u003e\u003cp\u003eWe constructed another donor vector CY7869-25 to target another functional gene \u003cem\u003efadD\u003c/em\u003e of \u003cem\u003eE. coli\u003c/em\u003e and then also detected expected sequence replacement in the target gene (Fig. S2D).\u003c/p\u003e\u003cp\u003eWe also constructed donor vector CY7869 in which, two MsDFID gdts targeting \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e gene \u003cem\u003edctA\u003c/em\u003e and \u003cem\u003earoA\u003c/em\u003e, respectively, were in tandem and fused to a single APE (Fig. S3). As a result, the APE in CY7869 can mediate simultaneous seamless sequence replacement in both of the two target regions (Fig. S3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.3\u003c/em\u003e. \u003cem\u003eIdentification of single clones of E. coli Top10 bearing APE-mediated sequence replacement in thyA\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThymidylate synthase (ThyA) in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e is involved in the \u003cem\u003ede novo\u003c/em\u003e synthesis of dTTP from dUMP. Without ThyA, the cell is unable to synthesize DNA and, therefore, will not grow in minimum growth media. Thus, \u003cem\u003ethyA\u003c/em\u003e-null \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e mutant can be selected (positive selection) by culture in growth medium in the absence of thymine, because \u003cem\u003ede novo\u003c/em\u003e dTTP synthesis can proceed without the need for \u003cem\u003eThyA\u003c/em\u003e function when thymine is provided (Wong et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Here, to further assess the efficiency of sequence replacement, we applied the selection system with ThyA as the selectable negative marker for selecting single colons of \u003cem\u003eE. coli\u003c/em\u003e with expected sequence replacement in \u003cem\u003ethyA\u003c/em\u003e gene as previously described (Wong et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). We designed fusion of APE and MsDFID gdt to target \u003cem\u003ethyA\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As a result, we found that pRiceSLthyA-3 which contains gdt-APE fusion can mediate precise sequence replacement in \u003cem\u003ethyA\u003c/em\u003e, while the negative vector, pnoSLthyA-3 with only MsDFID gdt, wasn\u0026rsquo;t able to achieve sequence replacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB-C and Fig. S4). However, the sequence replacement efficiency is relatively low (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). This observation further revealed that APE, can mediate precise sequence replacement.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.4\u003c/em\u003e. \u003cem\u003eGenetic requirements for gdt-guided sequence replacement in E. coli\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eNext, we sought to determine the genetic requirements for APE-mediate target sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. As above-mentioned, we selected \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strain TOP10, \u003cem\u003erecA-\u003c/em\u003eloss-of-function (\u003cem\u003erecA1\u003c/em\u003e) mutant, to exclude the possibility that endogenous RecA-dependent and independent HDR is responsible for the sequence replacement. TOP10 contains CRISPR-Cas3 system, which belongs to Type I CRISPR-Cas system and involves two protein elements for DNA targeting and cleavage, respectively: Cascade and Cas3; \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cascade is a multimeric complex of 5 different Cas proteins and responsible for processing CRISPR arrays and for binding target DNA sequences through PAM and protospacer recognition, whereas Cas3, the signature protein, is responsible for cleaving and degrading target DNA (Luo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The Cascade proteins include \u003cem\u003ecas8\u003c/em\u003e (\u003cem\u003ecse1\u003c/em\u003e), \u003cem\u003ecas11\u003c/em\u003e (\u003cem\u003ecse2\u003c/em\u003e), \u003cem\u003ecas7\u003c/em\u003e, \u003cem\u003ecas5\u003c/em\u003e (\u003cem\u003ecas5e\u003c/em\u003e) and \u003cem\u003ecas6e\u003c/em\u003e, and their open reading frames (ORFs) overlap with each other in the order of \u003cem\u003ecas8\u003c/em\u003e-\u003cem\u003ecas11\u003c/em\u003e-\u003cem\u003ecas7\u003c/em\u003e-\u003cem\u003ecas5\u003c/em\u003e-\u003cem\u003ecas6e\u003c/em\u003e, thus forming the Cascade operon under the control of \u003cem\u003ecas8\u003c/em\u003e promoter which is in downstream of \u003cem\u003ecas3\u003c/em\u003e gene expression cassette (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 system has been identified to mediate a long range and unidirectional genomic DNA deletion upstream of PAM without prominent off-target activity in eukaryotic cells (Luo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Li et al.,2018; Morisaka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The removal of Cas3 from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 system would allow Cascade to bind target DNA sequences but without subsequent degradation (Luo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). So, these insights lead us to wonder whether CRISPR-Cas3 system enables the endogenous APE-mediated sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e genome in this study. We first knocked out \u003cem\u003ecas3\u003c/em\u003e gene from TOP10 by deleting most of its genomic sequence to create loss-of-function mutant of \u003cem\u003ecas3\u003c/em\u003e, TOP10Δ\u003cem\u003ecas3\u003c/em\u003e (Fig. S5A). Then, we transformed both TOP10Δ\u003cem\u003ecas3\u003c/em\u003e and wild type TOP10 strain with one of above-mentioned donor vector F7869, and checked whether sequence replacement still happened in target region by sequencing genotyping PCR product (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Our results indicated no sequence replacement was observed in the transformed TOP10Δ\u003cem\u003ecas3\u003c/em\u003e strain, indicating that the APE-mediated sequence replacement is dependent on the endogenous \u003cem\u003ecas3\u003c/em\u003e gene. We then asked whether 5 Cascade proteins of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e are required for the desired sequence replacement in this study. Toward this aim, we simultaneously knocked out genes \u003cem\u003ecas8\u003c/em\u003e and \u003cem\u003ecas11\u003c/em\u003e, or \u003cem\u003ecas11\u003c/em\u003e, \u003cem\u003ecas7\u003c/em\u003e and \u003cem\u003ecas5\u003c/em\u003e, or \u003cem\u003ecas5\u003c/em\u003e and \u003cem\u003ecas6\u003c/em\u003e in a streamlining way, to generate multiplex knockout mutants of TOP10 strain, TOP10Δ\u003cem\u003ecas8cas11\u003c/em\u003e, TOP10Δ\u003cem\u003ecas11cas7cas5\u003c/em\u003e and TOP10Δ\u003cem\u003ecas5cas6e\u003c/em\u003e, respectively (Fig. S5B-D). Surprisingly, knockout of these \u003cem\u003ecas\u003c/em\u003e genes can\u0026rsquo;t abrogate desired sequence replacement event in all of these TOP10 mutant strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Thus, among \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 system, Cas3 might be the only Cas protein required for the APE-mediated targeted sequence replacement in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. Meanwhile, we used the same donor vector to transform \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains BL21 and BL21 (DE3) which are derived from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e B strain and inherently lacks CRISPR-Cas3 system (Fig. S6). As expected, we can\u0026rsquo;t detect the desired sequence replacement in target region in transformed BL21 and BL21 (DE3) strains (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB), consistent with the above observation of \u003cem\u003ecas3\u003c/em\u003e gene being required for the sequence replacement.\u003c/p\u003e\u003cp\u003eTherefore, these results revealed that endogenous Cas3 is the only genetic requirement for the gdt-mediated sequence replacement in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.5\u003c/em\u003e. \u003cem\u003eSpCas9 can rescue loss-of-function of E. coli Cas3 for seamless sequence replacement in E. coli\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eAs Cas9 and Cas3 share functional conservatism as shown in cleaving and degrading target DNA when engineered in genome editing (Luo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Morisaka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Cs\u0026ouml;rgő et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), we sought to test whether Cas9 protein functionally mimic Cas3 for the seamless sequence replacement. We used vector pCas9 with functional \u003cem\u003ecas9\u003c/em\u003e gene (\u003cem\u003eSpcas9\u003c/em\u003e) of \u003cem\u003eStreptococcus pyogenes\u003c/em\u003e Type II CRISPR-Cas system (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA) and donor vector F7869 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) to double-transform the above-used \u003cem\u003eE. coli\u003c/em\u003e strain TOP10Δ\u003cem\u003ecas3\u003c/em\u003e, loss-of-function mutant of Cas3. The genotyping PCR result demonstrated that SpCas9 protein can also be programmed to achieve precise sequence replacement in the target region of \u003cem\u003eE. coli\u003c/em\u003e as Cas3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and thus the mechanism of genome editing is conservative between \u003cem\u003eE. coli\u003c/em\u003e Cas3 and SpCas9.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e2.6\u003c/em\u003e. \u003cem\u003eThe sequence replacement via gdt is probably guided by RNA\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eFor all of the above donor vectors, sgdRNA, the fused structure of MsDFID gdt and APE, can be expressed into single RNA due to the activation by \u003cem\u003elacZ\u003c/em\u003e promoter in \u003cem\u003eE. coli\u003c/em\u003e. So, one important question to be settled in this context is the following: the genome edition in this study is guided by RNA or DNA directly, or both? So, we modified the above-used vectors F7869 by deleting the \u003cem\u003elacZ\u003c/em\u003e promoter, thus generating vectors F7869M with no virtual promoter to regulate the expression of the sgdRNA in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). After transformation of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e using F7869M, we detected no sgdRNA expression compared with that by F7869 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB), and consequently identified no sequence replacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). This reveals preliminarily that the sequence replacement strategy via fused gdt might mainly depend on RNA, but not DNA- guided, mechanism.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eHere we demonstrate that the simple tool by coupling MsDFID gdt and a newly identified gdt scaffold, APE, can realize precise sequence replacement. This strategy reveals feasibility of really PAMless and off-target-free genome editing. It promises to be particularly useful for installing substitutions, insertions and deletions, especially for accurate modification of genes into their alleles harboring allele-specific point mutations or seamless insertion of short regulatory elements to fine-tune gene expression. This will exhibit especial potential for broad applicability for therapy and basic research provided its efficiency would be further improved.\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.1\u003c/em\u003e. \u003cem\u003eThe MsDFID gdt-mediated sequence replacement in this study might well represent one solution towards PAM-independence and minimizing the risk of off-target due to its high reliability\u003c/em\u003e\u003c/p\u003e\u003cp\u003eDNA sequence replacement relies on delivery of a donor repair template (DRT) into the target cell for HDR of double-stranded DNA breaks (DSBs) which, in general, are repaired through either NHEJ or HDR. NHEJ is not precise and often causes random and therefore nonspecific InDels or other mutations at the junctions between target site and its flanking sequence (Lu et al., 2016). On the contrary, HDR is precise and can hence be used to achieve precise and sequence replacement for various kinds of gene modification. However, NHEJ is the predominant pathway, while HDR is relatively rare. In this study, all the targeted DNA insertion/deletion events were seamless and in the desired direction. On the other hand, high-throughput-sequencing data analysis pointed to no obvious genome-wide off-target in generating sequence replacement. This kind of precise and exclusive sequence replacement in all of target regions is in good correspondence with the known characteristics of HDR mechanism. These results implicate a dual role for MsDFID template as both guide RNA and donor template, therefore representing a new HDR mechanism for genome editing.\u003c/p\u003e\u003cp\u003eZuccaro et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) recently reported that, in editing human embryos using CRISPR-Cas9 system, about half of Cas9-induced double-strand break is microhomology-mediated end joining, and the most common repair outcome of the breaks remain unrepaired. This led to an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, both on-target and off-target cleavage of Cas9 results in frequent chromosome loss and hemizygous InDels because of cleavage of both alleles. These findings indicate that employing those widely-used gene edition tools would pose a substantial risk of off-target, while the strategy presented in this study feature reliability and might well represent one solution to this challenge.\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.2\u003c/em\u003e. \u003cem\u003eThe strategy of MsDFID gdt-APE fusion specifies genome modification mechanism of E. coli Cas3 or Cas9 proteins to HDR mode\u003c/em\u003e\u003c/p\u003e\u003cp\u003eThe main function of CRISPR-Cas system in bacteria and archaea is widely considered to protect prokaryotic cells from exogenous genetic materials, such as virus or plasmid. The structural and functional landscape of \u003cem\u003eE. coli\u003c/em\u003e Cascade complex (CRISPR-Cas3) characterized this system as RNA-guided immune surveillance system to defense viral invasion (Dutra et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Morisaka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhao et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In fact, type I-E CRISPR-Cas system which \u003cem\u003eE. coli\u003c/em\u003e CRISPR-Cas3 belongs to is the most extensively studied subtype. However, surprisingly perhaps, Bozic et al addressed involvement of this system in regulating endogenous genes at transcriptional level other than the canonical immune response (Bozic et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Thereby, relying more on its plausibility than on direct and convincing experimental proof, the notion of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 functioning as native defense system is circumstantial and a controversial subject so far (Mulepati et al., 2013;Sinkunas et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Here we provided \u003cem\u003ein vivo\u003c/em\u003e evidence in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e to implicate \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cas3 in targeted modification of endogenous genomic sequence in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, thereby shedding new light on those seemingly contradictory reports.\u003c/p\u003e\u003cp\u003eTaken together, the above data allow us to propose the model in which Cas3 or Cas9 activity could be readily reprogrammed by APE to achieve precise sequence replacement (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The APE could be recognized and bound by \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cas3 or Cas9 protein, thus forming MsDFID gdt/Cas3 or Cas9 ribonucleoprotein. Previous related reports revealed that \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e CRISPR-Cas3 system, but not Cas3 protein only, when being engineered to edit the genome of other organisms, is dependent on PAM and non-HDR DNA repair mode to generate large deletions (Luo et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Morisaka et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Dolan et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Alternatively, in this study, APE specifies genome modification mechanism of Cas3 protein to efficient HDR mode without limitation of PAM (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). And sgdRNA may function as a RNA donor repair template (DRT) for homology-directed DNA repair (HDR) of double-stranded DNA breaks, a transcript-templated HDR (TT-HDR).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003e3.3\u003c/em\u003e. \u003cem\u003eThe multiple DNA fragment insertion/deletion as donor sites in MsDFID template is a key genetic requirement for the sequence replacement\u003c/em\u003e\u003c/p\u003e\u003cp\u003eBoth crossover and non-crossover events involving chromatids during meiosis cause intragenic recombination that can generate new alleles or new combinations of existing alleles (Zhang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhou et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In fact, meiotic recombination is a major driver of genetic diversity, species evolution, and agricultural improvement. A higher rate of both intrachromosomal recombination (presumably via cross-over event) and mutation (mechanistically uncoupled) preferentially occur in high diversity domains in chromosomes during meiosis (Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Heterozygosity might well be causative for this finding, thus convincingly explaining the more diverse population-wide mutation rate variations including intragenic/allelic recombination in progeny of heterozygotes than homozygotes among F2 population (Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The underlying mechanism might be that heterozygosity would enhance the likelihood of poor pairing and consequently proceeding to Spo11-mediated DSBs for physically exposed regions (unsynapsed chromosomes and chromatin loops (Yang et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA three-stranded nucleic acid structure termed R-loops is universally formed during transcription, as the nascent RNA molecule may hybridize with the template DNA strand, forming a DNA:RNA hybrid and leaving the nontemplated DNA single-stranded (Yang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). R-loops have been found to persist throughout the genomes of various species (Yang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The common chromatin features of R-loop promote ectopic HR activity, thereby participating in a number of physiological processes, such as gene expression, DNA replication, and DNA and histone modifications, and DNA damage repair and genome stability (Yang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Sun et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). On the other hand, R-loops can lead to DNA damage as the single-strand DNA (ssDNA) formed from RNA/DNA hybridization is susceptible to mutagenes and lesions, leading to double-strand breaks and ectopic recombination that further induces genome instability (Yang et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eSimilarly, a sequential double-strained DNA (dsDNA) target searching and degradation process by Type I-E CRISPR Cascade of \u003cem\u003eThermobifida fusca\u003c/em\u003e (\u003cem\u003eTfuCascade\u003c/em\u003e) feature the unwinding of entire protospacer to form a full R-loop which triggers conformational changes in Cascade, licensing Cas3 to bind (Xiao et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In fact, in the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The structure provides the necessary temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The negative and positive checkpoints coordinately ensure stringent yet efficient target degradation in type I CRISPR-Cas systems (Xiao et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eFrom these knowledge, we suppose that in searching target dsDNA, the DNA fragment insertion/deletion array which features MsDFID gdt, could enhance the heterozygosity between gdt and its target strand. This would enable the formation of multiple bulges in gdt or its target strand (complementary strand) when they pairs with each other. Consequently, these bulges probably promote the priming of efficient HDR but not canonical NHEJ mode for sequence replacement by Cas3 or Cas9 proteins in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e. In other words, these bulges might well act as key rate-limiting factors to improve HDR efficiency and in turn HDR-mediated sequence replacement.\u003c/p\u003e\u003cp\u003eNotably, among the multiple DNA insertion/deletions (donor sites) in MsDFID gdt, the closer the one is to APE, the higher sequence replacement rate in the respective target site. So, for desired efficient sequence placement, DNA fragment insertion/deletion array need to be designed to form MsDFID gdt, but the donor site of interest should be arranged as the nearest to APE. This will achieve the sequence replacement only in its respective target site with the highest likelihood, thereby making the selection process as easy as possible.\u003c/p\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e3.4\u003c/em\u003e. \u003cem\u003eFunctional conservation between E. coli Cas3 and SpCas9\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe enormous diversity of viruses and their complex patterns of coevolution with defense systems suggests that more types of defense systems with diverse mechanisms can be expected to exist than are currently known (Pausch et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Gao et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In fact, in the context of an incessant arms race with mobile genetic elements, evolution of CRISPR-Cas systems has resulted in extreme diversification of both Cas protein sequences and architecture of the CRISPR-Cas loci (Shmakov et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given such an exciting forefront, considerable effort focused on exploring more CRISPR-Cas or CRISPR-Cas-like systems for providing more powerful addition to genome editing toolbox; whereas we devoted our efforts to dissecting and harnessing the cross-compatibility of different CRISPR-Cas systems.\u003c/p\u003e\u003cp\u003eObviously, the engineering of fusing MsDFID gdt to APE in this study is a kind of simplified strategy for genome editing. Although \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e Cas3 and SpCas9 proteins are derived from evolutionally divergent eubacteria, both of them can recognize the newly discovered gdt scaffold, APE. This revealed a kind of conservation in functional mechanism between the two Cas proteins which represent class 1 (type I) and class 2 systems (type II), respectively. Importantly, the knowledge will facilitate heterologous reconstitution of other well-characterized Cas proteins and MsDFID gdt to establish HDR as only/main genome edition mechanism for reprogrammable sequence replacement.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Materials and methodology","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e4.1\u003c/em\u003e. \u003cem\u003eUsed E. coli strains\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eThe used \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains are listed in Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e4.2\u003c/em\u003e. \u003cem\u003eE. coli growth conditions\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eFor strain and plasmid construction, \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains were cultured in Luria-Bertani (LB) medium except for special requirement. For fermentation, strains were cultured in 50 mL M9 medium containing 2 g/L Amicase (Sigma, St. Louis, MO, USA), 0.2 g/L l-arabinose, and 1% (v/v) glycerol; 3% glycerol was used for PHB production. To maintain the plasmids, final concentrations of 100 \u0026micro;g/mL ampicillin, 50 \u0026micro;g/mL spectinomycin, and 25 \u0026micro;g/mL chloromycetin were added to the corresponding cultures.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e4.3\u003c/em\u003e. \u003cem\u003eConstruction of sgdRNA expression vectors (donor vectors)\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eFused gdRNA and scaffold APE, sgdRNAs, were synthesized and cloned into pUC57 vector, thereby yielding donor plasmid F7869, pRiceSLthyA-3 and other expression vectors. For F7869M, fused gdRNA and scaffold APE was cloned into pUC57m (modified pUC57 vector) without actual promoter near the multi-cloning site (MCS).\u003c/p\u003e\u003cp\u003eThe sequences of target region and donor sites regarding F7869/F7869M are listed in Table S2.\u003c/p\u003e\u003cp\u003epCas9 vector with a stable expression system of functional SpCas9 in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, was purchased from Addgene, a non-profit plasmid repository.\u003c/p\u003e\u003cp\u003e\u003cem\u003e4.4\u003c/em\u003e. \u003cem\u003eApplication of ThyA selection system for identifying single clones of E. coli bearing efficient and seamless DNA sequence replacement in thyA\u003c/em\u003e\u003c/p\u003e\u003cp\u003eTwo \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strains, TOP10 and TOP10Δ\u003cem\u003ecas3\u003c/em\u003e, loss-of-function mutant of \u003cem\u003ecas3\u003c/em\u003e of TOP10, were used in this experiment. Cells were grown in a modified M9 minimal medium as previously described, containing 48 mM Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e7H\u003csub\u003e2\u003c/sub\u003eO, 22 mM KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e, 8.6 mM NaCl and 18.7 mM NH\u003csub\u003e4\u003c/sub\u003eCl, 1 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.5 mM CaCl\u003csub\u003e2\u003c/sub\u003e, 0.4% (w/v) glucose, 0.6 mM leucine, valine and isoleucine, 30 nM vitamin B1, 1% (v/v) Luria-Bertani (LB) broth and 1\u0026times;MM1 containing 1 nM each of ZnSO\u003csub\u003e4\u003c/sub\u003e, CuSO\u003csub\u003e4\u003c/sub\u003e, Na\u003csub\u003e2\u003c/sub\u003eMoO\u003csub\u003e4\u003c/sub\u003e, CoCl\u003csub\u003e2\u003c/sub\u003e, MnSO\u003csub\u003e4\u003c/sub\u003e, CrCl\u003csub\u003e3\u003c/sub\u003e and NiCl\u003csub\u003e2\u003c/sub\u003e (Wong et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Specific growth selection medium was supplemented with either or combination of 50 mg/ml ampicillin, 12.5 mg/ml chloramphenicol, 0.79 mM thymine (Sigma) and 10 mg/ml (for plasmids) trimethoprim (Sigma). For growth on agar plates, 2.5% (w/v) noble agar (Sigma) was included in the appropriate medium. All chemicals were of analytical grade.\u003c/p\u003e\u003cp\u003eAppropriate \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e cells were prepared for electroporation as described previously (Wong et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In brief, 50 ml of \u0026lsquo;competent\u0026rsquo; cells were mixed with 2\u0026ndash;8 mg of plasmids in a pre-cooled 0.1 cm Gene Pulser cuvette (Bio-Rad Laboatories) and electroporated using the Bio-Rad Gene Pulser with conditions set at 1.8 kV, 25 mF and 200 ohms. Following electroporation, the cells were grown in 1 ml of LB broth at 37\u0026deg;C for 1-1.5 h, collected by centrifugation and plated at various dilutions onto M9 minimal medium agar plates with appropriate supplements for selective growth, and incubated at 37\u0026deg;C for 48\u0026ndash;72 h.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e4.5\u003c/em\u003e. \u003cem\u003eIdentification of sequence replacement in target region\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eGenomic DNA from cell pellets of single clones of transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e was extracted using Ezup Column Bacteria Genomic DNA Purification Kit according to the manufacturer\u0026rsquo;s protocol (Sangon Biotech Co. Ltd., Shanghai, China). Genomic PCR was performed using Green Taq Mix containing High-Fidelity Taq DNA polymerase DNA、dNTP and optimized buffer (Q5) according to the manufacturer\u0026rsquo;s protocol (Vazyme Co. Ltd., Nanjing, China). To genotype the types of sequence replacement in target regions, the PCR products were purified and then subjected to direct DNA Sanger-sequencing service, or ligated into pUC18-T T-vector for transformation of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e and then Sanger-sequencing plasmid.\u003c/p\u003e\u003cp\u003eAll of used primers are listed in Supplementary Table S4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003e4.6\u003c/em\u003e. \u003cem\u003eHigh-throughput sequencing of genomic DNA and analysis\u003c/em\u003e\u003c/h2\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e\u003cem\u003e4.6.1.\u003c/em\u003eTotal DNA extraction and quality control\u003c/h2\u003e\u003cp\u003eTotal genome DNA from transformed \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e by donor vectors was extracted using CTAB/SDS method. The integrity of DNA was measured by using 1% gel electrophoresis. DNA concentration and purity were measured by the NanoDrop 200 Spectrophotometer (Thermo Fisher, USA), and OD260/280 was 1.8-2.0. DNA concentration was measured by Qubit 3.0 Fluorometer (Life Technologies, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e4.6.2. Library preparation\u003c/h2\u003e\u003cp\u003eDNA sequencing libraries were generated using the KAPA-HyperPlus Kit according to the manufacturer\u0026rsquo;s protocol (The extracted DNA was broken into fragments, then terminal repair, 3' terminal addition, ligation, and addition of primers, perform size selection, PCR amplification and purification, and quality inspection of every library.). Sequencing was performed by the Bioacme Biotechnology Co., Ltd. (Wuhan, China). The resulting libraries were size-checked by using an Agilent 2100 Bioanalyzer system (Agilent). Finally, the library preparations were sequenced on an Illumina NovaSeq platform and 150 bp paired-end reads were generated.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e4.6.3. Data analysis\u003c/h2\u003e\u003cp\u003eRaw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality. Reference genome and gene model annotation files were downloaded from \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e genome K-12. Clean reads were aligned to the reference genome using short read mapping software BWA. As a result, approximately 99.99% of the reference genome was covered with uniquely mapping, non-duplicate reads, to an average coverage depth of around 200X. Reads were mapped onto the reference genome and single nucleotide polymorphisms (SNPs) or InDels were detected by BCFtools software. The SVs (Structural Variations) were identified using DELLY2. The copy number variations of every sample were using CNVnator software. The detected variant sites were annotated using ANNOVAR.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e4.7. Knockout of E. coli Cas proteins\u003c/h2\u003e\u003cp\u003eThe knockout of \u003cem\u003ecas3\u003c/em\u003e, \u003cem\u003ecas8-cas11\u003c/em\u003e, \u003cem\u003ecas11-cas7-cas5\u003c/em\u003e and \u003cem\u003ecas5-cas6e\u003c/em\u003e in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e strain TOP10 was conducted by using coupled CRISPR/Cas9 technology and λ Red homologous recombination system as previously described (Jiang et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The resulting loss-of-function mutant strains, TOP10Δ\u003cem\u003ecas3\u003c/em\u003e, TOP10Δ\u003cem\u003ecas5cas6e\u003c/em\u003e, TOP10Δ\u003cem\u003ecas8cas11\u003c/em\u003e and TOP10Δ\u003cem\u003ecas11cas7cas5\u003c/em\u003e were verified by PCR and agarose gel electrophoresis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e4.8. RNA extraction and semi-quantitative RT-PCR\u003c/h2\u003e\u003cp\u003eTotal RNA was extracted from plant tissues using TRIzol reagent (Life Technologies) and treated with RNase-free DNase I (Invitrogen). Two micrograms of RNA were reverse transcribed using oligo (dT) primer and AMV reverse transcriptase (Promega). Quantitative PCR experiments were performed. Each qRT-PCR assay was replicated at least three times with three independent RNA preparations.\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e 16S rRNA was used as internal control. Primers used are listed in Supplementary Table S4.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003e4.9. Statistics\u003c/h2\u003e\u003cp\u003eAn unpaired two-sided Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was implemented for group comparison as indicated in the Fig. legends.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRui Li\u003c/strong\u003e: Writing-review \u0026amp; editing, Writing-original draft, Data curation, Formal analysis, Resources. \u003cstrong\u003eZeyu Qi\u003c/strong\u003e: Writing-original draft, Formal analysis, Data curation, Conceptualization, Methodology. \u003cstrong\u003eYueyang Wanghuang\u003c/strong\u003e: Data curation, Formal analysis, Resources.\u0026nbsp;\u003cstrong\u003eYonghan Xu\u003c/strong\u003e: Writing-review \u0026amp; editing, Writing-original draft, Methodology, Funding acquisition, Investigation, Data curation, Conceptualization, Project administration, Supervision. \u003cstrong\u003eDechuan Wu\u003c/strong\u003e: Writing-original draft, Methodology, Data curation.\u0026nbsp;\u003cstrong\u003eWei Tang\u003c/strong\u003e. Software, Methodology, Formal analysis, Data curation.\u0026nbsp;\u003cstrong\u003eXiang Chen\u003c/strong\u003e: Data curation, Investigation, Methodology.\u0026nbsp;\u003cstrong\u003eXiaoyu Hu\u003c/strong\u003e: Conceptualization, Data curation, Methodology, Resources.\u0026nbsp;\u003cstrong\u003eGuoxing Zou\u003c/strong\u003e: Investigation, Resources, Writing- review \u0026amp; editing.\u0026nbsp;\u003cstrong\u003eJiahao Hua\u003c/strong\u003e: Data curation, Formal analysis, Methodology, Resources.\u0026nbsp;\u003cstrong\u003eZhangying Feng\u003c/strong\u003e: Data curation, Formal analysis, Methodology, Resources.\u003cstrong\u003eXuelan Zong\u003c/strong\u003e: Data curation, Formal analysis, Methodology, Resources.\u003cstrong\u003eQiutao Hu\u003c/strong\u003e: Data curation, Formal analysis, Methodology, Resources.\u0026nbsp;\u003cstrong\u003eJingcai Li\u003c/strong\u003e: Conceptualization, Funding acquisition, Project administration, Resources, Supervision.\u0026nbsp;\u003cstrong\u003eXinchun Lin\u003c/strong\u003e:\u0026nbsp;Conceptualization,\u0026nbsp;Funding acquisition, Project administration, Resources, Supervision, Writing-original draft, Writing-review \u0026amp; editing.\u003cstrong\u003eXiaobo Wang\u003c/strong\u003e:\u0026nbsp;Conceptualization, Project administration, Resources, Supervision,\u0026nbsp;Writing-review \u0026amp; editing. \u003cstrong\u003eChuanxi Ma\u003c/strong\u003e:\u0026nbsp;Conceptualization, Funding acquisition, Project administration, Resources, Supervision.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthical approval and consent to participate\u003c/p\u003e\n\u003cp\u003eNot applicable. This article does not include any studies conducted by the authors involving human participants or animals. All research and analyses presented are based on experimental methods and data that do not include human or animal subjects. This disclaimer ensures compliance with ethical standards and regulations regarding research involving human and animal participants.\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests. The views and opinions expressed in this article are purely those of the authors and do not necessarily reflect the official policy or position of author's employer or any Novartis officers.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability Data will be made available on request.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Prof. Yang Sheng for technical support help in developing \u003cem\u003eThyA\u003c/em\u003e selection system to identify single clones of \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e bearing seamless DNA sequence replacement in\u003cem\u003e\u0026nbsp;thyA,\u003c/em\u003e Prof. Yongshen Liu for support in experiment design technical support and Prof. Songhu Wang for critical reading of the manuscript and discussion. This work is supported by University Collaborative Creation Project of Anhui Province to C.X.M. (grant no. GXXT-2019-033). Y.H.X. was supported by Anhui Provincial Nature Science Foundation (No. 1808085MC87), a startup\u0026nbsp;fund\u0026nbsp;(No. 2018008) from Anhui Agricultural University, and Industry Alliance Project of Dabie Mountain Experimental Station of Anhui Agricultural University (No. 202408). W. T. \u0026nbsp;was supported National Natural Science Foundation of China (No. 31700266).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData and materials availability\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll data, including the sequencing data of genotyping PCR products and the data related to the high-throughput sequencing of genomic DNA and analysis, are available from corresponding author C.X.M. upon request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAnzalone A, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, Chen PJ, Wilson C, Newby GA, Raguram A, et al. Search-and- replace genome editing without double-strand breaks or donor DNA. Nature. 2019;576:149\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBozic B, Repac J, Djordjevic M. 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Cell. 2020;183:1650\u0026ndash;64.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"AT-rich pincer-like elements (APE), gdt/Cas3 or gdt/Cas9 ribonucleoprotein, genome editing, sequence replacement","lastPublishedDoi":"10.21203/rs.3.rs-7640593/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7640593/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSequence replacement is the most direct and powerful genome editing strategy. However, it remains currently an urgent need to develop protospacer adjacent motif (PAM)-less, off-target-free and simple tools for precise sequence replacement. To address this challenge, we fused a rice-derived AT-rich pincer-like elements (APE), which is composed of unique repeat-spacer array, to donor template against target sequence, thus forming guider and donor template (gdt). The donor template harbors multiple sites of DNA fragment insertion/deletion (MsDFID) which function as donor sites. APE plays as MsDFID gdt scaffold to repurpose Cas3 or Cas9 to mediate transposition of DNA fragment insertion/deletion from MsDFID donor template into genome target in \u003cem\u003eE\u003c/em\u003e. \u003cem\u003ecoli\u003c/em\u003e, thus realizing seamless sequence replacement. These results established putative gdt/Cas3 or gdt/Cas9 ribonucleoprotein as compact genome editors which feature PAM-lessness, no observable off-target, and simplicity based on the dual role of MsDFID gdt \u003cem\u003eper se\u003c/em\u003e as both guider and donor template. This strategy provides significant potential for precise sequence replacement in both animals and plants.\u003c/p\u003e","manuscriptTitle":"PAMless and precise sequence replacement by gdt/Cas3 or gdt/Cas9 ribonucleoprotein","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-23 13:45:03","doi":"10.21203/rs.3.rs-7640593/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cfcd16ee-8b4f-461a-a122-2f9eb1b96d0f","owner":[],"postedDate":"October 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-29T06:39:06+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-23 13:45:03","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7640593","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7640593","identity":"rs-7640593","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}