Enhancing the HoSeI method for one-step large-scale genome integration in Escherichia coli using multi-cutter plasmids

Enhancing the HoSeI method for one-step large-scale genome integration in Escherichia coli using multi-cutter plasmids | 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 Article Enhancing the HoSeI method for one-step large-scale genome integration in Escherichia coli using multi-cutter plasmids Riho Hirano, Hayato Fujita, Ayane Minagawa, Kaneyoshi Yamamoto This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9262883/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Precise chromosomal integration of large DNA fragments remains a significant challenge in metabolic engineering of Escherichia coli, largely due to the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs). To overcome this issue, we have developed a modular and versatile "multi-cutters" platform comprising plasmids designed to express up to four distinct sgRNAs simultaneously from a single vector. Building upon the HoSeI (Homologous Sequence Integration) method, this platform synergizes CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair to enable one-step, scarless editing. Our construction strategy uses a modular assembly approach with specific linkers and restriction sites to enable the rapid and flexible generation of double, triple and quadruple cutters. We demonstrate the utility of this platform by successfully 'rewriting' genes—the one-step replacement of approximately 1 kbp chromosomal regions—by replacing the high-expression ompC and ompF loci with fluorescent reporter genes (mCherry and gfp). The resulting dual-reporter strain, RHA00802, functioned as an effective osmotic biosensor, exhibiting concentration-dependent fluorescence responses to sucrose levels (0–10%) in accordance with the regulation of the EnvZ–OmpR two-component system. This multi-cutter platform and gene rewriting strategy provides a powerful, generalized toolkit for complex genomic design and the rapid construction of functional microbial devices. (198 words) Biological sciences/Biological techniques Biological sciences/Biotechnology Biological sciences/Molecular biology Escherichia coli HoSeI method Multi-cutter plasmid Gene rewriting Biosensor Figures Figure 1 Figure 2 Figure 3 Introduction Genome editing is an indispensable technology for altering biological functions and serves as a cornerstone of synthetic biology and metabolic engineering (1,2). Escherichia coli is the main microbial platform for these fields and is extensively employed in the production of biofuels, amino acids and high-value polymer precursors, such as 1,4-butanediol (3-5). However, the complex metabolic modulation required for industrial-scale production necessitates the use of efficient genome editing tools that are capable of precise, sequential or multiplexed manipulations (1,2,6). Traditional genome engineering in E. coli has long relied on λ-Red-mediated recombineering (1,6,7). However, these methods often depend on selectable markers, which require additional steps for removal and frequently leave behind undesirable genomic "scars" (e.g., FRT or loxP sites) (1,7-10). Although scarless methods have been developed, they often involve laborious, multi-step procedures, such as the 'insertion-then-removal' of dual-selectable markers, which significantly limit overall throughput (2,7,10-12). Integration of the CRISPR-Cas9 system has transformed this field by providing a programmable mechanism for creating targeted double-strand breaks (DSBs), effectively killing unedited cells and eliminating the need for selectable markers (1). Although CRISPR-based tools are widely used for point mutations and small deletions, the single-step integration of large DNA fragments (over 1 kb), which we refer to as 'gene rewriting', remains technically challenging (1,10,13,14). One major issue is the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs) (2,15,16). Even within the same gene, different target loci can result in a difference in editing success by a factor of a hundred (17). To overcome these limitations, our laboratory previously developed the HoSeI (Homologous Sequence Integration) method (18). This one-step, scarless technique combines CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair. It utilises a pCas plasmid and a specialised sgRNA expression plasmid (genome cutter) to induce DSBs (6,18) (see Fig. 1). While the HoSeI method has been successfully used for iterative functional deletions, realizing its full potential for gene rewriting requires a more robust strategy to ensure genome cleavage at any target locus (Fig. 1). In this study, we present a modular, versatile platform for 'multi-cutters' —plasmids designed to express up to four distinct sgRNAs simultaneously (Fig. 1). We demonstrate the utility of this platform by successfully rewiring approximately 1 kbp regions of the genome, specifically replacing the high-expression ompC and ompF loci with fluorescent reporter genes ( mCherry and GFP ). This advancement provides a powerful toolkit for complex genomic design, enabling the rapid construction of functional biosensors and the optimisation of metabolic pathways in E. coli . Results and Discussion The construction of "multi-cutters" expressing multiple sgRNA sequences on a single plasmid To overcome the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs), we developed a modular platform for constructing "multi-cutters"—plasmids capable of expressing up to four distinct sgRNA sequences simultaneously. This strategy, illustrated in Figure 2, utilizes a modular assembly approach where multiple sgRNA units are integrated in a tandem arrangement. 1) Construction of Double Cutters We first established the feasibility of constructing double cutters through multiple cloning techniques. Using In-Fusion cloning, we joined a vector fragment (containing sgRNA–yfeH3) and an insert fragment (containing sgRNA–yfeH4) to generate psgRNA–yfeH3–yfeH4. Verification by agarose gel electrophoresis and Sanger sequencing confirmed that all of six examined clones contained both sequences exactly as designed. Alternatively, we demonstrated that double cutters could be derived from existing triple cutters through restriction enzyme treatment. By utilizing the Not Ⅰ sites strategically placed within the modular linkers, the second sgRNA module was excised, followed by self-ligation. This allowed for the rapid generation of diverse double-cutter variants listed in Table 2, such as psgRNA-rpoS5–lacI7 and psgRNA-lacI7–rpoF2, all of which were confirmed by size analysis via electrophoresis. 2) Modular Assembly of Triple Cutters using Gibson Assembly The platform was further expanded to triple cutters using Gibson Assembly to increase the number of genomic targets. Our design utilized single-cutter plasmids as templates to amplify sgRNA modules with primers that added 5' linker sequences containing mutually distinguishable restriction sites, including Nco Ⅰ, Not Ⅰ, Sph Ⅰ, and Mlu Ⅰ. To verify this modular system, we targeted the lacI , rpoS , and rpoF genes. By directionally assembling a vector fragment and two insert fragments, we successfully constructed various tandem arrangements, such as psgRNA-lacI7-rpoS5-rpoF2. The presence of all three modules was confirmed by Not Ⅰ digestion, which yielded the expected 0.9 kbp and 3.5 kbp fragments, and further validated by Sanger sequencing. 3) Expansion to Quadruple Cutters and Functional Validation Finally, we extended the platform to build quadruple cutters to facilitate the replacement of entire open reading frames (ORFs), a process we term "gene rewriting". As shown in Table 2, we targeted high-expression loci such as ompC and ompF . For the ompC locus, four sgRNAs (ompC4, 18, 38, and 51) were selected and assembled into a single vector (psgRNA-ompC4-ompC18-ompC38-ompC51) via Gibson Assembly. The functionality of these quadruple cutters was validated by measuring their transformation efficiency into E. coli MG1655 harboring the pCas plasmid. The transformation efficiencies of both the psgRNA-ompC4-ompC18-ompC38-ompC51 and the psgRNA-ompF46-ompF16-ompF33-ompF48 were 0% compared to the psgRNA vector control, while the ompC - and ompF -single cutters showed relative transformation efficiencies of 63.8–0%, indicating robust and synergistic genome cleavage at multiple sites within the target region. This established multi-cutter platform provided the essential technical foundation for achieving successful gene rewriting, enabling the one-step replacement of the ~1 kbp ompC and ompF genes with fluorescent reporter sequences ( mCherry and gfp ) to construct functional biosensors. Gene rewriting of ompF and ompC loci for the construction of an osmotic biosensor To achieve the one-step replacement of chromosomal ORFs with fluorescent reporter genes—a process we define as "gene rewriting"—we utilized the established HoSeI method combined with newly developed quadruple cutters. We first aimed to replace the approximately 1 kbp ompF region with the green fluorescent protein gene ( gfp ) (Table 1). The single quadruple-cutter plasmid, psgRNA-ompF46-ompF16-ompF33-ompF48 (Table 2), was used as a genome cutter. The linear dsDNA genome editor for ompF was prepared by PCR using pDiGc as a template, featuring 130-bp homology arms flanking the gfp sequence. Cotransformation of the quadruple cutter and the genome editor into recombineering-proficient cells yielded several ampicillin-resistant colonies. Colony PCR screening identified a clone with the expected 1,200-bp size shift corresponding to gfp integration, which was further validated by Sanger sequencing and designated as strain KMA00106 ( ΔompF::gfp ) (Fig. 3a). Following a similar strategy, we targeted the ompC locus for replacement with the red fluorescent protein gene ( mCherry ). We utilized a quadruple cutter, psgRNA-ompC4-ompC18-ompC38-ompC51 (Table 2). The genome editor was prepared using pmCherry-N1 as a template with primers designed to provide homology to the ompC flanking regions. Genome editing of MG1655 resulted in the isolation of strain AMA00107 ( ΔompC::mCherry ) (Table 1), confirmed by the detection of an 800-bp PCR product. To construct a functional dual-reporter biosensor, sequential gene rewriting was performed by introducing the mCherry editing machinery into the KMA00106 ( ΔompF::gfp ) background (Fig. 3a). Transformants were screened via colony PCR, identifying multiple clones that exhibited the 800-bp mCherry amplicon alongside the established gfp integration. The resulting dual-reporter strain, carrying both ΔompF::gfp and ΔompC::mCherry , was designated as RHA00802 (Table 1). The ability of strain RHA00802 to respond to osmotic stress was evaluated by measuring fluorescence intensities under varying concentrations of sucrose (0%, 5%, and 10%) using a SpectraMax iD3 plate reader. In accordance with the transcriptional regulation of the EnvZ–OmpR two-component system (19), we observed a concentration-dependent decrease in GFP fluorescence (driven by the ompF promoter) and a corresponding concentration-dependent increase in mCherry fluorescence (driven by the ompC promoter) (Fig. 3b). To date, we have constructed a comprehensive library of over 1,300 genome cutters, providing strong evidence for the robustness of the multi-cutter platform. These results demonstrate that our multi-cutter platform and the HoSeI method enable the precise and efficient rewriting of genetic regions into functional biological tools. Methods Construction of multi-cutter plasmids To construct multi-cutter plasmids capable of expressing multiple sgRNAs, specific DNA fragments were prepared and assembled as follows. The vector fragment containing the replication origin (pMB1 ori) and the ampicillin resistance gene ( bla ) was amplified using KOD-Plus-Neo (Toyobo, Japan) with psgRNA as a template. A 50 μL reaction mixture containing 1 U/μL polymerase, 1× PCR buffer, 0.2 mM dNTPs, 1.5 mM MgSO 4 , 0.3 μM each of primers Primer-V-L1 and Primer-V-U2, and 5 ng of a psgRNA plasmid as template was subjected to initial denaturation at 95°C for 30 s, followed by 18 cycles of 95°C for 30 s, 55°C for 1 min, and 75°C for 3 min using a MiniAmp Thermal Cycler (Thermo Fisher Scientific, USA). Insert fragments containing specific sgRNA sequences were amplified using KOD-Plus-Neo with corresponding single-cutter plasmids as templates. The primers were designed to include 5' linker sequences and unique restriction sites (NcoI, NotI, SphI, or MluI) to facilitate directional assembly (Table 3). The PCR conditions consisted of an initial denaturation at 94°C for 2 min, followed by 30 cycles of 98°C for 10 s and 68°C for 90 s. All PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio, Japan) and eluted in 15 μL of elution buffer. Double-cutter plasmids were assembled using the In-Fusion HD Cloning kit (Takara Bio). Briefly, 0.5 μL of purified vector DNA, 1 pmol of insert DNA, and 2 μL of 5× In-Fusion HD Enzyme Premix were reacted at 50°C for 15 min. Triple- and quadruple-cutter plasmids were assembled using Gibson Assembly Master Mix (New England Biolabs, USA). A 20 μL reaction mixture containing 10 μL of Master Mix, 0.06 pmol of vector fragment, and 0.03 pmol of each insert fragment was incubated at 50°C for 15 min. The assembly products were transformed into E. coli DH5-α or NEB 5-alpha competent cells. After heat shock at 42°C for 30–45 s and recovery in SOC medium for 1 h at 37°C, the cells were plated on LB agar containing 100 μg/mL ampicillin and incubated overnight at 37°C. Transformants were cultured overnight in LB liquid medium with ampicillin, and plasmids were extracted using the Plasmid DNA Extraction Mini Kit (Favorgen, Taiwan). The construction of multi-cutters was initially verified by agarose gel electrophoresis to confirm the expected increase in plasmid size compared to the single-cutter vector. The identity and arrangement of the sgRNA sequences were confirmed by Sanger sequencing using a 3500 Genetic Analyzer (Applied Biosystems, USA) with BigDye Terminator v1.1 or v3.1 cycle sequencing kits. Specific sequencing primers (Seq-primer-1 to 4) complementary to the linker regions were utilized to verify the insertion of each sgRNA module. Sequencing data were analyzed to ensure that all sgRNA sequences and their respective promoters were correctly integrated as designed Preparation of genome editor Linear double-stranded DNA (dsDNA) genome editors were prepared via PCR to facilitate the replacement of target chromosomal ORFs with fluorescent reporter genes. For rewriting the ompF locus, the gfp gene was amplified from the plasmid pDiGc (Helaine et al., 2010) using primers gfp_in_ompF_F and gfp_in_ompF_R (Table 3). To target the ompC locus, the mCherry gene was amplified from the pmCherry-N1 vector (Takara Bio, Japan) using primers mCherry_in_ompC_F and mCherry_in_ompC_R (Table 3). Each 160-nucleotide primer was designed with a modular structure: the 3' terminal 30 bp were complementary to the fluorescent protein gene, while the 5' terminal 130 bp provided homology arms corresponding to the upstream or downstream flanking regions of the target genomic locus. PCR was performed in 50 μL reaction mixtures containing 1 U/μL KOD-Plus-Neo polymerase (Toyobo, Japan), 1× PCR buffer, 0.2 mM dNTPs, 1.5 mM MgSO 4 , 0.3 μM of each primer, and 5 ng of template plasmid. The thermal cycling profile consisted of an initial denaturation at 95°C for 30 s, followed by 18 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 75°C for 3 min using a MiniAmp Thermal Cycler (Thermo Fisher Scientific, USA). All PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio) and eluted in 15 μL of elution buffer. DNA concentration and purity were determined by spectrophotometry, and the target fragments were verified by agarose gel electrophoresis. Genome editing and gene rewriting Gene rewriting, such as the replacement of genomic ORFs with fluorescent reporter genes, was performed using the HoSeI method by combining multi-cutter plasmids and linear dsDNA genome editors. To prepare recombineering-proficient cells, a single colony of E. coli MG1655 harboring the pCas plasmid was cultured overnight in LB medium containing 50 μg/mL kanamycin. The culture was diluted 100-fold into fresh LB medium supplemented with 50 μg/mL kanamycin and 0.2% (w/v) L-arabinose to induce the expression of the λ-Red recombinase (Exo, Beta, Gam) encoded by pCas. Cells were grown to an OD 660 of 0.2–0.3 and then harvested by centrifugation at 5,000 × g for 10 min at 4°C. The cell pellet was washed and resuspended in ice-cold 50 mM CaCl 2 , followed by incubation on ice for 60 min to achieve competency. For long-term storage, the cells were resuspended in 50 mM CaCl 2 containing 15% (v/v) glycerol, aliquoted into 100 μL volumes, and stored at -80°C. For the genome editing reaction, 100 μL of pCas-harboring competent cells were thawed on ice and cotransformed with 100–250 ng of a multi-cutter plasmid and 100–500 ng of a linear dsDNA genome editor. After incubation on ice for 30 min, the cells were subjected to heat shock at 42°C for 45 s. The cells were immediately recovered in 900 μL of SOC medium supplemented with 1% (w/v) L-arabinose and incubated for 1–3 h at 30°C to allow for λ-Red-mediated homologous recombination and repair of the Cas9-induced double-strand breaks. Following recovery, the cells were harvested by centrifugation, resuspended in a small volume of the supernatant, and plated on LB agar containing ampicillin and kanamycin. Successful gene rewriting was verified by colony PCR using Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific) with specific checking primers (ompF-check-F/R or ompC-check-F/R) (Table 3). The thermal cycling profile consisted of initial denaturation at 95°C for 30 s, followed by 18 cycles of 98°C for 30 s, 60°C for 10 s, and 75°C for 15 s. The PCR products were analyzed by 1% agarose gel electrophoresis to confirm the expected size shifts (e.g., 1.2 kbp for gfp , 0.8 kbp for mCherry , 1.1 kbp for ompC , and 1.2 kbp for ompF ). Targeted regions were further purified using the NucleoSpin Gel and PCR Clean-up kit and validated by Sanger sequencing using BigDye Terminator v3.1 and a 3500 Genetic Analyzer (Applied Biosystems). To obtain plasmid-free mutant strains, the genome cutter and pCas plasmids were successively eliminated. First, the genome cutter was removed by culturing the edited strains in LB medium containing 50 μg/mL kanamycin and 1 mM IPTG at 30°C overnight. IPTG induces the expression of sgRNA-pMB1 from the pCas plasmid, which targets the replication origin of the genome cutter, leading to its degradation. Colonies were screened by replica plating to select for ampicillin-sensitive clones. Subsequently, the pCas plasmid was cured by culturing the selected clones in LB medium at 42°C overnight. Because pCas possesses a temperature-sensitive origin of replication ( repA101 ts ), it becomes unable to replicate at high temperatures. Kanamycin-sensitive colonies were then isolated as verified, plasmid-free, scar-less genome-edited strains. Fluorescence measurements of GFP and mCherry To quantify the expression of fluorescent reporter genes, single colonies were picked from agar plates and grown overnight in 3 mL of LB liquid medium. Subcultures were prepared by inoculating 100 μL of the overnight culture into 10 mL of fresh LB medium and incubating with shaking at 37°C until the OD 660 reached 0.2–0.5. Cells were harvested by centrifugation at 1,200 rpm for 10 min and washed twice with 1 mL of sterile water. The resulting cell pellets were resuspended in 500 μL of sterile water, and 200 μL of the suspension was transferred to a 96-well plate. Optical density at 660 nm and fluorescence intensities were measured using a SpectraMax iD3 plate reader (Molecular Devices). GFP fluorescence was detected at an excitation wavelength of 478 nm and an emission wavelength of 519 nm, while mCherry fluorescence was detected at excitation and emission wavelengths of 577 nm and 620 nm, respectively. All measurements were performed in three independent biological replicates (n=3). For data analysis, the average OD 660 and fluorescence values from wells containing only sterile water were used as background and subtracted from each sample measurement to calculate the net OD 660 and net fluorescence. The final fluorescence value for each strain was defined as the ratio of net fluorescence to net OD 660 . Results are presented as the mean ± standard deviation from the triplicate experiments. Declarations Acknowledgement We thank Daiki Ono and Kanon Murano of Hosei University for establishing the set of genome cutter. Funding This work was supported by a Grant-in-Aid for Scientific Research (C) [20K05795 and 23K04998] from MEXT. Data Availability The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The E. coli strains and plasmids will be provided from NBRP of Japan. Author contributions R.H., H.F., A.M., K.M., and K.Y. performed the experiments and analyzed the data; K.Y. conceived the study and wrote the paper. Competing Interests The authors declare no competing interests. References Dong, H., Cui, Y. & Zhang, D. CRISPR/Cas Technologies and their applications in Escherichia coli . Front. Bioeng. Biotechnol. 9 , 762676 (2022). Lim, S. R. & Lee, S. J. Multiplex CRISPR-Cas genome editing: next-generation microbial strainengineering. J. Agric. Food Chem. 72 , 11871–11884 (2024). Yan, M. Y. et al. CRISPR-Cas12a-assisted recombineering in bacteria. Appl. Environ. Microbiol. 83 , e00947-17 (2017). Yan, W. X. et al. Functionally diverse Type V CRISPR-Cas systems. Science 363 , 88–91 (2019). Yang, D. et al. Escherichia coli as a platform microbial host for systems metabolic engineering. Essays Biochem . 65 , 225–246 (2021). Jiang, Y. et al. Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system. Appl. Environ. Microbiol. 81 , 2506–2514 (2015). Reisch, C. R. & Prather, K. L. J. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in Escherichia coli . Sci. Rep. 5 , 15096 (2015). Datsenko, K. A. & Wanner, B. L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. U. S. A. 97 , 6640–6645 (2000). Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol . 2 , 2006.0008 (2006). Bassalo, M. C. et al. Rapid and efficient one-step metabolic pathway integration in E. coli . ACS Synth. Biol. 5 , 561–568 (2016). Stringer, A. M. et al. FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica . PLOS ONE 7 , e44841 (2012). Kim, J., Webb, A. M., Kershner, J. P., Blaskowski, S. & Copley, S. D. A versatile and highly efficient method for scarless genome editing in Escherichia coli and Salmonella enterica . BMC Biotechnol. 14 , 84 (2014). Wu, M. Y. et al. Combining CRISPR and CRISPRi systems for metabolic engineering of E. coli and 1,4-BDO biosynthesis. ACS Synth. Biol. 6 , 2350–2361 (2017). Ronda, C., Pedersen, L. E., Sommer, M. O. & Nielsen, A. T. CRMAGE: CRISPR optimized MAGE recombineering. Sci. Rep. 6 , 19452 (2016). Charpentier, E. & Schroeder, R. RNA techniques for bacteria. Curr. Opin. Microbiol. 10 , 254–256 (2007). Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154 , 442–451 (2013). Bryant, J. A., Sellars, L. E., Busby, S. J. & Lee, D. J. Chromosome position effects on gene expression in Escherichia coli K-12. Nucleic Acids Res. 42 , 11383–11392 (2014). Miyake, Y. & Yamamoto, K. Epistatic effect of regulators to the adaptive growth of Escherichia coli . Sci. Rep . 10 , 3661 (2020). Kenney, L. J. & Anand, G. S. EnvZ/OmpR Two-component signaling: an archetype system that can function noncanonically. EcoSal Plus 9 , ESP-0001-2019 (2020). Hayashi, K. et al. Highly accurate genome sequences of Escherichia coli K-12 strains MG1655 and W3110. Mol. Syst. Biol. 2 , 2006.0007 (2006). Helaine, S. et al. Dynamics of intracellular bacterial replication at the single cell level. Proc. Natl. Acad. Sci. U. S. A. 107 , 3746–3751 (2010). Tables Tables 1 to 3 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 16 Apr, 2026 Reviews received at journal 16 Apr, 2026 Reviews received at journal 12 Apr, 2026 Reviewers agreed at journal 11 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 03 Apr, 2026 Editor invited by journal 03 Apr, 2026 Editor assigned by journal 01 Apr, 2026 Submission checks completed at journal 01 Apr, 2026 First submitted to journal 30 Mar, 2026 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9262883","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":618970464,"identity":"aa2374c1-413f-4eaa-a9f8-f5cac64699fb","order_by":0,"name":"Riho Hirano","email":"","orcid":"","institution":"Hosei University","correspondingAuthor":false,"prefix":"","firstName":"Riho","middleName":"","lastName":"Hirano","suffix":""},{"id":618970465,"identity":"9089200b-dfd7-4e6b-9dcb-fe13c28f92c3","order_by":1,"name":"Hayato Fujita","email":"","orcid":"","institution":"Hosei University","correspondingAuthor":false,"prefix":"","firstName":"Hayato","middleName":"","lastName":"Fujita","suffix":""},{"id":618970466,"identity":"e3c9313e-bf85-45d6-8e21-15bcf669953e","order_by":2,"name":"Ayane Minagawa","email":"","orcid":"","institution":"Hosei University","correspondingAuthor":false,"prefix":"","firstName":"Ayane","middleName":"","lastName":"Minagawa","suffix":""},{"id":618970467,"identity":"099101ad-5cbf-4180-944c-7d3544b6ebfc","order_by":3,"name":"Kaneyoshi Yamamoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA60lEQVRIiWNgGAWjYBACCRiDH8wyALMTiNMiOYNkLQY3iHWYZPvZYxI/c+yijW83H/7woYBBnr+B4dkDfFqkefLSJHu3Jeduu3MsTXKGAYPhjAMM6Qb4tMgx5JhJ8G5jzt12I8eMmceAgXEDA0OaBF4t/G/MJP9uq8/dPCPH+PMfAwZ7glqkJXLMpHm3Hc7dIJFjIA0MsUSCWiRnvDG2lt12PHfGjbQ0yR4DieQZhwn4ReJ8juHNt9uqc/tnJB/+8OOPjW1/e0/aA3xagIAF2RlANjNPGgEdDMwf0ATYjxHSMgpGwSgYBSMLAAADLkZKsrSgswAAAABJRU5ErkJggg==","orcid":"","institution":"Hosei University","correspondingAuthor":true,"prefix":"","firstName":"Kaneyoshi","middleName":"","lastName":"Yamamoto","suffix":""}],"badges":[],"createdAt":"2026-03-30 06:09:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9262883/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9262883/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106725346,"identity":"368ae918-ec1a-474e-8001-a9579bac3e40","added_by":"auto","created_at":"2026-04-12 18:32:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1269684,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGene rewriting in \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eE. coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e genome via HoSeI method.\u003c/strong\u003e [a] Genome editing HoSeI method is performed via CRISPR-Cas9-mediated cleavage using an sgRNA expression plasmid (genome cutter) and repair through the λRed homologous recombination reaction using dsDNA (genome editor). By culturing the cells in a medium containing IPTG, sgRNA-pMB1—which targets the replication origin of the genome cutter—is expressed from pCas, leading to the removal of the sgRNA expression plasmid from the E. coli cells. Furthermore, by culturing at 42°C, pCas (which possesses a temperature-sensitive origin of replication) becomes unable to replicate and is no longer maintained in the cells, allowing for the isolation of plasmid-free genome-edited strains. [b] In conventional HoSeI methods, only about a dozen nucleotides could be edited at once. Therefore, to edit a long region, rather than using a standard genome cutter that cleaves only one site, this study developed a \"quadruple cutter\" that cleaves four locations within a target gene region simultaneously and verified its effectiveness. Since each sgRNA recognizes a different position on the target gene, the desired mutants can be obtained with significantly less time and effort than before.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9262883/v1/739869f7b4d110984eada166.png"},{"id":106481889,"identity":"050907a3-1e19-41f9-ae81-d08f46a0b9a7","added_by":"auto","created_at":"2026-04-09 05:04:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":169957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eModular Design Concept for construction of a multi-cutter.\u003c/strong\u003e [a] This panel illustrates the modular assembly strategy for constructing multi-cutters using abstract units designated as sgRNA-A, B, C, and D. Each sgRNA unit is amplified via PCR using specific primer sets designed to add 5' linker sequences and mutually distinguishable restriction enzyme recognition sites, including NcoⅠ, NotⅠ, SphⅠ, and MluⅠ.The vector fragment is designed to encompass the origin of replication (pMB1 ori) and the antibiotic resistance gene (\u003cem\u003ebla\u003c/em\u003e), while the insert fragments are prepared to amplify only the specific sgRNA regions. A key feature of this design is the emphasis on the homologous overlap regions created between the vector and insert fragments, which enables seamless and directional connection through Gibson Assembly or In-Fusion cloning. By utilizing these overlapping sequences, multiple sgRNA modules can be integrated into a single plasmid in a precise, tandem arrangement, a double-cutter (a), a triple-cutter (b), and quadruple-cutter (c).\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9262883/v1/10146e0e8c14c4d509bcf681.png"},{"id":106481891,"identity":"928a06b1-c854-48d2-ac3b-0fb4513951f4","added_by":"auto","created_at":"2026-04-09 05:04:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":639160,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLarge-scale gene rewriting for the construction of an osmotic biosensor using the HoSeI method.\u003c/strong\u003e [a] Schematic of the \u003cem\u003eompF\u003c/em\u003e locus rewriting: To replace the ~1 kbp \u003cem\u003eompF\u003c/em\u003e gene with the green fluorescent protein gene (\u003cem\u003egfp\u003c/em\u003e), a quadruple cutter plasmid expressing four specific sgRNAs (sgRNA-ompF46, 16, 33, 46 and 48) was utilized to induce multiple double-strand breaks across the target ORF. A linear dsDNA genome editor containing the \u003cem\u003egfp\u003c/em\u003e sequence flanked by homology arms was introduced to facilitate scar-less integration via λ-Red-mediated homologous recombination. Next, the \u003cem\u003eompC\u003c/em\u003e gene was targeted for replacement with the red fluorescent protein gene (\u003cem\u003emCherry\u003c/em\u003e) using a quadruple cutter carrying sgRNA-ompC4, 18, 38, and 51. Through sequential editing, the dual-reporter strain RHA00802 (\u003cem\u003eΔompF::gfp, ΔompC::mCherry\u003c/em\u003e) was established. [b] Functional validation of the osmotic sensor: The graph displays the fluorescence intensities of GFP (green circles) and mCherry (red squares) in the RHA00802 strain cultured under varying osmotic pressures (0–10% sucrose). Fluorescence values are normalized by the optical density at 660 nm (fluorescence/ OD\u003csub\u003e660\u003c/sub\u003e). In accordance with the EnvZ–OmpR two-component system regulation, GFP expression (driven by the \u003cem\u003eompF\u003c/em\u003e promoter) decreases while mCherry expression (driven by the \u003cem\u003eompC\u003c/em\u003e promoter) increases as the sucrose concentration rises, demonstrating the successful rewriting of genomic loci into a functional biosensor. Error bars represent the standard deviation from three independent biological replicates (n=3).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9262883/v1/35b621f587c91914fda2b1aa.png"},{"id":106726993,"identity":"e9a5629b-95fe-435b-a975-851173b81b38","added_by":"auto","created_at":"2026-04-12 18:37:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2630379,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9262883/v1/ca2283cd-50a1-4bf3-9aa3-1dab718fc49d.pdf"},{"id":106724550,"identity":"8e122dab-df9b-482c-87e9-4eb2ebc858b0","added_by":"auto","created_at":"2026-04-12 18:28:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18647,"visible":true,"origin":"","legend":"","description":"","filename":"Tables.docx","url":"https://assets-eu.researchsquare.com/files/rs-9262883/v1/01a45fdd47c664f73e667d35.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing the HoSeI method for one-step large-scale genome integration in Escherichia coli using multi-cutter plasmids","fulltext":[{"header":"Introduction","content":"\u003cp\u003eGenome editing is an indispensable technology for altering biological functions and serves as a cornerstone of synthetic biology and metabolic engineering (1,2). \u003cem\u003eEscherichia coli\u003c/em\u003e is the main microbial platform for these fields and is extensively employed in the production of biofuels, amino acids and high-value polymer precursors, such as 1,4-butanediol (3-5). However, the complex metabolic modulation required for industrial-scale production necessitates the use of efficient genome editing tools that are capable of precise, sequential or multiplexed manipulations (1,2,6). Traditional genome engineering in \u003cem\u003eE. coli\u003c/em\u003e has long relied on λ-Red-mediated recombineering (1,6,7). However, these methods often depend on selectable markers, which require additional steps for removal and frequently leave behind undesirable genomic \"scars\" (e.g., \u003cem\u003eFRT\u003c/em\u003e or \u003cem\u003eloxP\u003c/em\u003e sites) (1,7-10). Although scarless methods have been developed, they often involve laborious, multi-step procedures, such as the 'insertion-then-removal' of dual-selectable markers, which significantly limit overall throughput (2,7,10-12).\u0026nbsp;Integration of the CRISPR-Cas9 system has transformed this field by providing a programmable mechanism for creating targeted double-strand breaks (DSBs), effectively killing unedited cells and eliminating the need for selectable markers (1).\u003c/p\u003e\n\u003cp\u003eAlthough CRISPR-based tools are widely used for point mutations and small deletions, the single-step integration of large DNA fragments (over 1 kb), which we refer to as 'gene rewriting', remains technically challenging (1,10,13,14). One major issue is the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs) (2,15,16).\u0026nbsp;Even within the same gene, different target loci can result in a difference in editing success by a factor of a hundred (17).\u0026nbsp;To overcome these limitations, our laboratory previously developed the HoSeI (Homologous Sequence Integration) method\u0026nbsp;(18). This one-step, scarless technique combines CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair. It utilises a pCas plasmid and a specialised sgRNA expression plasmid (genome cutter) to induce DSBs (6,18) (see Fig. 1).\u003c/p\u003e\n\u003cp\u003eWhile the HoSeI method has been successfully used for iterative functional deletions, realizing its full potential for gene rewriting requires a more robust strategy to ensure genome cleavage at any target locus (Fig. 1). In this study, we present a modular, versatile platform for 'multi-cutters' —plasmids designed to express up to four distinct sgRNAs simultaneously (Fig. 1). We demonstrate the utility of this platform by successfully rewiring approximately 1 kbp regions of the genome, specifically replacing the high-expression \u003cem\u003eompC\u003c/em\u003e and \u003cem\u003eompF\u003c/em\u003e loci with fluorescent reporter genes (\u003cem\u003emCherry\u003c/em\u003e and \u003cem\u003eGFP\u003c/em\u003e). This advancement provides a powerful toolkit for complex genomic design, enabling the rapid construction of functional biosensors and the optimisation of metabolic pathways in \u003cem\u003eE. coli\u003c/em\u003e.\u003c/p\u003e"},{"header":" Results and Discussion","content":"\u003cp\u003e\u003cstrong\u003eThe construction of \"multi-cutters\" expressing multiple sgRNA sequences on a single plasmid\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo overcome the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs), we developed a modular platform for constructing \"multi-cutters\"—plasmids capable of expressing up to four distinct sgRNA sequences simultaneously. This strategy, illustrated in Figure 2, utilizes a modular assembly approach where multiple sgRNA units are integrated in a tandem arrangement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1) Construction of Double Cutters\u003c/strong\u003e We first established the feasibility of constructing double cutters through multiple cloning techniques. Using In-Fusion cloning, we joined a vector fragment (containing sgRNA–yfeH3) and an insert fragment (containing sgRNA–yfeH4) to generate psgRNA–yfeH3–yfeH4. Verification by agarose gel electrophoresis and Sanger sequencing confirmed that all of six examined clones contained both sequences exactly as designed. Alternatively, we demonstrated that double cutters could be derived from existing triple cutters through restriction enzyme treatment. By utilizing the \u003cem\u003eNot\u003c/em\u003eⅠ\u0026nbsp;sites strategically placed within the modular linkers, the second sgRNA module was excised, followed by self-ligation. This allowed for the rapid generation of diverse double-cutter variants listed in Table 2, such as psgRNA-rpoS5–lacI7 and psgRNA-lacI7–rpoF2, all of which were confirmed by size analysis via electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2) Modular Assembly of Triple Cutters using Gibson Assembly\u003c/strong\u003e The platform was further expanded to triple cutters using Gibson Assembly to increase the number of genomic targets. Our design utilized single-cutter plasmids as templates to amplify sgRNA modules with primers that added 5' linker sequences containing mutually distinguishable restriction sites, including \u003cem\u003eNco\u003c/em\u003eⅠ, \u003cem\u003eNot\u003c/em\u003eⅠ, \u003cem\u003eSph\u003c/em\u003eⅠ, and \u003cem\u003eMlu\u003c/em\u003eⅠ. To verify this modular system, we targeted the \u003cem\u003elacI\u003c/em\u003e, \u003cem\u003erpoS\u003c/em\u003e, and \u003cem\u003erpoF\u003c/em\u003e genes. By directionally assembling a vector fragment and two insert fragments, we successfully constructed various tandem arrangements, such as psgRNA-lacI7-rpoS5-rpoF2. The presence of all three modules was confirmed by \u003cem\u003eNot\u003c/em\u003eⅠ\u0026nbsp;digestion, which yielded the expected 0.9 kbp and 3.5 kbp fragments, and further validated by Sanger sequencing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3) Expansion to Quadruple Cutters and Functional Validation\u003c/strong\u003e Finally, we extended the platform to build quadruple cutters to facilitate the replacement of entire open reading frames (ORFs), a process we term \"gene rewriting\". As shown in Table 2, we targeted high-expression loci such as \u003cem\u003eompC\u003c/em\u003e and \u003cem\u003eompF\u003c/em\u003e. For the \u003cem\u003eompC\u003c/em\u003e locus, four sgRNAs (ompC4, 18, 38, and 51) were selected and assembled into a single vector (psgRNA-ompC4-ompC18-ompC38-ompC51) via Gibson Assembly. The functionality of these quadruple cutters was validated by measuring their transformation efficiency into \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eMG1655 harboring the pCas plasmid. The transformation efficiencies of both the psgRNA-ompC4-ompC18-ompC38-ompC51 and the psgRNA-ompF46-ompF16-ompF33-ompF48 were 0% compared to the psgRNA vector control, while the \u003cem\u003eompC\u003c/em\u003e- and \u003cem\u003eompF\u003c/em\u003e-single cutters showed relative transformation efficiencies of 63.8–0%, indicating robust and synergistic genome cleavage at multiple sites within the target region. This established multi-cutter platform provided the essential technical foundation for achieving successful gene rewriting, enabling the one-step replacement of the ~1 kbp \u003cem\u003eompC\u003c/em\u003e and \u003cem\u003eompF\u003c/em\u003e genes with fluorescent reporter sequences (\u003cem\u003emCherry\u003c/em\u003e and \u003cem\u003egfp\u003c/em\u003e) to construct functional biosensors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGene rewriting of \u003cem\u003eompF\u003c/em\u003e and \u003cem\u003eompC\u003c/em\u003e loci for the construction of an osmotic biosensor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo achieve the one-step replacement of chromosomal ORFs with fluorescent reporter genes—a process we define as \"gene rewriting\"—we utilized the established HoSeI method combined with newly developed quadruple cutters.\u003c/p\u003e\n\u003cp\u003eWe first aimed to replace the approximately 1 kbp \u003cem\u003eompF\u003c/em\u003e region with the green fluorescent protein gene (\u003cem\u003egfp\u003c/em\u003e) (Table 1). The single quadruple-cutter plasmid, psgRNA-ompF46-ompF16-ompF33-ompF48 (Table 2), was used as a genome cutter. The linear dsDNA genome editor for ompF was prepared by PCR using pDiGc as a template, featuring 130-bp homology arms flanking the \u003cem\u003egfp\u003c/em\u003e sequence. Cotransformation of the quadruple cutter and the genome editor into recombineering-proficient cells yielded several ampicillin-resistant colonies. Colony PCR screening identified a clone with the expected 1,200-bp size shift corresponding to \u003cem\u003egfp\u003c/em\u003e integration, which was further validated by Sanger sequencing and designated as strain KMA00106 (\u003cem\u003eΔompF::gfp\u003c/em\u003e) (Fig. 3a). Following a similar strategy, we targeted the \u003cem\u003eompC\u003c/em\u003e locus for replacement with the red fluorescent protein gene (\u003cem\u003emCherry\u003c/em\u003e). We utilized a quadruple cutter, psgRNA-ompC4-ompC18-ompC38-ompC51 (Table 2). The genome editor was prepared using pmCherry-N1 as a template with primers designed to provide homology to the \u003cem\u003eompC\u003c/em\u003e flanking regions. Genome editing of MG1655 resulted in the isolation of strain AMA00107 (\u003cem\u003eΔompC::mCherry\u003c/em\u003e) (Table 1), confirmed by the detection of an 800-bp PCR product. To construct a functional dual-reporter biosensor, sequential gene rewriting was performed by introducing the mCherry editing machinery into the KMA00106 (\u003cem\u003eΔompF::gfp\u003c/em\u003e) background (Fig. 3a). Transformants were screened via colony PCR, identifying multiple clones that exhibited the 800-bp \u003cem\u003emCherry\u003c/em\u003e amplicon alongside the established \u003cem\u003egfp\u003c/em\u003e integration. The resulting dual-reporter strain, carrying both \u003cem\u003eΔompF::gfp\u003c/em\u003e and \u003cem\u003eΔompC::mCherry\u003c/em\u003e, was designated as RHA00802 (Table 1). The ability of strain RHA00802 to respond to osmotic stress was evaluated by measuring fluorescence intensities under varying concentrations of sucrose (0%, 5%, and 10%) using a SpectraMax iD3 plate reader. In accordance with the transcriptional regulation of the EnvZ–OmpR two-component system (19), we observed a concentration-dependent decrease in GFP fluorescence (driven by the \u003cem\u003eompF\u003c/em\u003e promoter) and a corresponding concentration-dependent increase in mCherry fluorescence (driven by the \u003cem\u003eompC\u003c/em\u003e promoter) (Fig. 3b). To date, we have constructed a comprehensive library of over 1,300 genome cutters, providing strong evidence for the robustness of the multi-cutter platform. These results demonstrate that our multi-cutter platform and the HoSeI method enable the precise and efficient rewriting of genetic regions into functional biological tools.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cstrong\u003eConstruction of multi-cutter plasmids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo construct multi-cutter plasmids capable of expressing multiple sgRNAs, specific DNA fragments were prepared and assembled as follows. The vector fragment containing the replication origin (pMB1 ori) and the ampicillin resistance gene (\u003cem\u003ebla\u003c/em\u003e) was amplified using KOD-Plus-Neo (Toyobo, Japan) with psgRNA as a template. A 50 μL reaction mixture containing 1 U/μL polymerase, 1× PCR buffer, 0.2 mM dNTPs, 1.5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.3 μM each of primers Primer-V-L1 and Primer-V-U2, and 5 ng of a psgRNA plasmid as template was subjected to initial denaturation at 95°C for 30 s, followed by 18 cycles of 95°C for 30 s, 55°C for 1 min, and 75°C for 3 min using a MiniAmp Thermal Cycler (Thermo Fisher Scientific, USA). Insert fragments containing specific sgRNA sequences were amplified using KOD-Plus-Neo with corresponding single-cutter plasmids as templates. The primers were designed to include 5' linker sequences and unique restriction sites (NcoI, NotI, SphI, or MluI) to facilitate directional assembly (Table 3). The PCR conditions consisted of an initial denaturation at 94°C for 2 min, followed by 30 cycles of 98°C for 10 s and 68°C for 90 s. All PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio, Japan) and eluted in 15 μL of elution buffer. Double-cutter plasmids were assembled using the In-Fusion HD Cloning kit (Takara Bio). Briefly, 0.5 μL of purified vector DNA, 1 pmol of insert DNA, and 2 μL of 5× In-Fusion HD Enzyme Premix were reacted at 50°C for 15 min. Triple- and quadruple-cutter plasmids were assembled using Gibson Assembly Master Mix (New England Biolabs, USA). A 20 μL reaction mixture containing 10 μL of Master Mix, 0.06 pmol of vector fragment, and 0.03 pmol of each insert fragment was incubated at 50°C for 15 min. The assembly products were transformed into \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eDH5-α or NEB 5-alpha competent cells. After heat shock at 42°C for 30–45 s and recovery in SOC medium for 1 h at 37°C, the cells were plated on LB agar containing 100 μg/mL ampicillin and incubated overnight at 37°C. Transformants were cultured overnight in LB liquid medium with ampicillin, and plasmids were extracted using the Plasmid DNA Extraction Mini Kit (Favorgen, Taiwan). The construction of multi-cutters was initially verified by agarose gel electrophoresis to confirm the expected increase in plasmid size compared to the single-cutter vector. The identity and arrangement of the sgRNA sequences were confirmed by Sanger sequencing using a 3500 Genetic Analyzer (Applied Biosystems, USA) with BigDye Terminator v1.1 or v3.1 cycle sequencing kits. Specific sequencing primers (Seq-primer-1 to 4) complementary to the linker regions were utilized to verify the insertion of each sgRNA module. Sequencing data were analyzed to ensure that all sgRNA sequences and their respective promoters were correctly integrated as designed\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of genome editor\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLinear double-stranded DNA (dsDNA) genome editors were prepared via PCR to facilitate the replacement of target chromosomal ORFs with fluorescent reporter genes. For rewriting the \u003cem\u003eompF\u003c/em\u003e locus, the \u003cem\u003egfp\u003c/em\u003e gene was amplified from the plasmid pDiGc (Helaine et al., 2010) using primers gfp_in_ompF_F and gfp_in_ompF_R (Table 3). To target the \u003cem\u003eompC\u003c/em\u003e locus, the \u003cem\u003emCherry\u003c/em\u003e gene was amplified from the pmCherry-N1 vector (Takara Bio, Japan) using primers mCherry_in_ompC_F and mCherry_in_ompC_R (Table 3). Each 160-nucleotide primer was designed with a modular structure: the 3' terminal 30 bp were complementary to the fluorescent protein gene, while the 5' terminal 130 bp provided homology arms corresponding to the upstream or downstream flanking regions of the target genomic locus. PCR was performed in 50 μL reaction mixtures containing 1 U/μL KOD-Plus-Neo polymerase (Toyobo, Japan), 1× PCR buffer, 0.2 mM dNTPs, 1.5 mM MgSO\u003csub\u003e4\u003c/sub\u003e, 0.3 μM of each primer, and 5 ng of template plasmid. The thermal cycling profile consisted of an initial denaturation at 95°C for 30 s, followed by 18 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, and extension at 75°C for 3 min using a MiniAmp Thermal Cycler (Thermo Fisher Scientific, USA). All PCR products were purified using the NucleoSpin Gel and PCR Clean-up kit (Takara Bio) and eluted in 15 μL of elution buffer. DNA concentration and purity were determined by spectrophotometry, and the target fragments were verified by agarose gel electrophoresis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eGenome editing and gene rewriting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGene rewriting, such as the replacement of genomic ORFs with fluorescent reporter genes, was performed using the HoSeI method by combining multi-cutter plasmids and linear dsDNA genome editors. To prepare recombineering-proficient cells, a single colony of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eMG1655 harboring the pCas plasmid was cultured overnight in LB medium containing 50 μg/mL kanamycin. The culture was diluted 100-fold into fresh LB medium supplemented with 50 μg/mL kanamycin and 0.2% (w/v) L-arabinose to induce the expression of the λ-Red recombinase (Exo, Beta, Gam) encoded by pCas. Cells were grown to an OD\u003csub\u003e660\u003c/sub\u003e of 0.2–0.3 and then harvested by centrifugation at 5,000 × g for 10 min at 4°C. The cell pellet was washed and resuspended in ice-cold 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e, followed by incubation on ice for 60 min to achieve competency. For long-term storage, the cells were resuspended in 50 mM CaCl\u003csub\u003e2\u003c/sub\u003e containing 15% (v/v) glycerol, aliquoted into 100 μL volumes, and stored at -80°C. For the genome editing reaction, 100 μL of pCas-harboring competent cells were thawed on ice and cotransformed with 100–250 ng of a multi-cutter plasmid and 100–500 ng of a linear dsDNA genome editor. After incubation on ice for 30 min, the cells were subjected to heat shock at 42°C for 45 s. The cells were immediately recovered in 900 μL of SOC medium supplemented with 1% (w/v) L-arabinose and incubated for 1–3 h at 30°C to allow for λ-Red-mediated homologous recombination and repair of the Cas9-induced double-strand breaks. Following recovery, the cells were harvested by centrifugation, resuspended in a small volume of the supernatant, and plated on LB agar containing ampicillin and kanamycin. Successful gene rewriting was verified by colony PCR using Platinum SuperFi II Green PCR Master Mix (Thermo Fisher Scientific) with specific checking primers (ompF-check-F/R or ompC-check-F/R) (Table 3). The thermal cycling profile consisted of initial denaturation at 95°C for 30 s, followed by 18 cycles of 98°C for 30 s, 60°C for 10 s, and 75°C for 15 s. The PCR products were analyzed by 1% agarose gel electrophoresis to confirm the expected size shifts (e.g., 1.2 kbp for \u003cem\u003egfp\u003c/em\u003e, 0.8 kbp for \u003cem\u003emCherry\u003c/em\u003e, 1.1 kbp for \u003cem\u003eompC\u003c/em\u003e, and 1.2 kbp for \u003cem\u003eompF\u003c/em\u003e). Targeted regions were further purified using the NucleoSpin Gel and PCR Clean-up kit and validated by Sanger sequencing using BigDye Terminator v3.1 and a 3500 Genetic Analyzer (Applied Biosystems). To obtain plasmid-free mutant strains, the genome cutter and pCas plasmids were successively eliminated. First, the genome cutter was removed by culturing the edited strains in LB medium containing 50 μg/mL kanamycin and 1 mM IPTG at 30°C overnight. IPTG induces the expression of sgRNA-pMB1 from the pCas plasmid, which targets the replication origin of the genome cutter, leading to its degradation. Colonies were screened by replica plating to select for ampicillin-sensitive clones. Subsequently, the pCas plasmid was cured by culturing the selected clones in LB medium at 42°C overnight. Because pCas possesses a temperature-sensitive origin of replication (\u003cem\u003erepA101\u003c/em\u003e\u003csup\u003ets\u003c/sup\u003e), it becomes unable to replicate at high temperatures. Kanamycin-sensitive colonies were then isolated as verified, plasmid-free, scar-less genome-edited strains.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFluorescence measurements of GFP and mCherry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo quantify the expression of fluorescent reporter genes, single colonies were picked from agar plates and grown overnight in 3 mL of LB liquid medium. Subcultures were prepared by inoculating 100 μL of the overnight culture into 10 mL of fresh LB medium and incubating with shaking at 37°C until the OD\u003csub\u003e660\u003c/sub\u003e reached 0.2–0.5. Cells were harvested by centrifugation at 1,200 rpm for 10 min and washed twice with 1 mL of sterile water. The resulting cell pellets were resuspended in 500 μL of sterile water, and 200 μL of the suspension was transferred to a 96-well plate. Optical density at 660 nm and fluorescence intensities were measured using a SpectraMax iD3 plate reader (Molecular Devices). GFP fluorescence was detected at an excitation wavelength of 478 nm and an emission wavelength of 519 nm, while mCherry fluorescence was detected at excitation and emission wavelengths of 577 nm and 620 nm, respectively. All measurements were performed in three independent biological replicates (n=3). For data analysis, the average OD\u003csub\u003e660\u003c/sub\u003e and fluorescence values from wells containing only sterile water were used as background and subtracted from each sample measurement to calculate the net OD\u003csub\u003e660\u003c/sub\u003e and net fluorescence. The final fluorescence value for each strain was defined as the ratio of net fluorescence to net OD\u003csub\u003e660\u003c/sub\u003e. Results are presented as the mean ± standard deviation from the triplicate experiments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Daiki Ono and Kanon Murano of Hosei University for establishing the set of genome cutter.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by a Grant-in-Aid for Scientific Research (C) [20K05795 and 23K04998] from MEXT.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request. The E. coli strains and plasmids will be provided from NBRP of Japan.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR.H., H.F., A.M., K.M., and K.Y. performed the experiments and analyzed the data; K.Y. conceived the study and wrote the paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eDong, H., Cui, Y. \u0026amp; Zhang, D. CRISPR/Cas Technologies and their applications in \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eFront. Bioeng. Biotechnol.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e9\u003c/strong\u003e, 762676 (2022).\u003c/li\u003e\n \u003cli\u003eLim, S. R. \u0026amp; Lee, S. J. Multiplex CRISPR-Cas genome editing: next-generation microbial strainengineering. \u003cem\u003eJ. Agric. Food Chem.\u003c/em\u003e \u003cstrong\u003e72\u003c/strong\u003e, 11871–11884 (2024).\u003c/li\u003e\n \u003cli\u003eYan, M. Y. et al. CRISPR-Cas12a-assisted recombineering in bacteria. \u003cem\u003eAppl. Environ. Microbiol.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e83\u003c/strong\u003e, e00947-17 (2017).\u003c/li\u003e\n \u003cli\u003eYan, W. X. et al. Functionally diverse Type V CRISPR-Cas systems. \u003cem\u003eScience\u003c/em\u003e \u003cstrong\u003e363\u003c/strong\u003e, 88–91 (2019).\u003c/li\u003e\n \u003cli\u003eYang, D. et al. \u003cem\u003eEscherichia coli\u003c/em\u003e as a platform microbial host for systems metabolic engineering. \u003cem\u003eEssays Biochem\u003c/em\u003e. \u003cstrong\u003e65\u003c/strong\u003e, 225–246 (2021).\u003c/li\u003e\n \u003cli\u003eJiang, Y. et al. Multigene editing in the \u003cem\u003eEscherichia coli\u003c/em\u003e genome via the CRISPR-Cas9 system. \u003cem\u003eAppl. Environ. Microbiol.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e81\u003c/strong\u003e, 2506–2514 (2015).\u003c/li\u003e\n \u003cli\u003eReisch, C. R. \u0026amp; Prather, K. L. J. The no-SCAR (Scarless Cas9 Assisted Recombineering) system for genome editing in \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 15096 (2015).\u003c/li\u003e\n \u003cli\u003eDatsenko, K. A. \u0026amp; Wanner, B. L. One-step inactivation of chromosomal genes in \u003cem\u003eEscherichia coli\u003c/em\u003e K-12 using PCR products. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u003c/em\u003e \u003cstrong\u003e97\u003c/strong\u003e, 6640–6645 (2000).\u003c/li\u003e\n \u003cli\u003eBaba, T. et al. Construction of \u003cem\u003eEscherichia coli\u003c/em\u003e K-12 in-frame, single-gene knockout mutants: the Keio collection. \u003cem\u003eMol. Syst. Biol\u003c/em\u003e. \u003cstrong\u003e2\u003c/strong\u003e, 2006.0008 (2006).\u003c/li\u003e\n \u003cli\u003eBassalo, M. C. et al. Rapid and efficient one-step metabolic pathway integration in \u003cem\u003eE. coli\u003c/em\u003e. \u003cem\u003eACS Synth. Biol.\u003c/em\u003e \u003cstrong\u003e5\u003c/strong\u003e, 561–568 (2016).\u003c/li\u003e\n \u003cli\u003eStringer, A. M. et al. FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in \u003cem\u003eEscherichia coli\u003c/em\u003e and \u003cem\u003eSalmonella enterica\u003c/em\u003e. \u003cem\u003ePLOS ONE\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e7\u003c/strong\u003e, e44841 (2012).\u003c/li\u003e\n \u003cli\u003eKim, J., Webb, A. M., Kershner, J. P., Blaskowski, S. \u0026amp; Copley, S. D. A versatile and highly efficient method for scarless genome editing in \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eand \u003cem\u003eSalmonella enterica\u003c/em\u003e. \u003cem\u003eBMC Biotechnol.\u003c/em\u003e \u003cstrong\u003e14\u003c/strong\u003e, 84 (2014).\u003c/li\u003e\n \u003cli\u003eWu, M. Y. et al. Combining CRISPR and CRISPRi systems for metabolic engineering of \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003eand 1,4-BDO biosynthesis. \u003cem\u003eACS Synth. Biol.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 2350–2361 (2017).\u003c/li\u003e\n \u003cli\u003eRonda, C., Pedersen, L. E., Sommer, M. O. \u0026amp; Nielsen, A. T. CRMAGE: CRISPR optimized MAGE recombineering. \u003cem\u003eSci. Rep.\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e, 19452 (2016).\u003c/li\u003e\n \u003cli\u003eCharpentier, E. \u0026amp; Schroeder, R. RNA techniques for bacteria. \u003cem\u003eCurr. Opin. Microbiol.\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, 254–256 (2007).\u003c/li\u003e\n \u003cli\u003eGilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. \u003cem\u003eCell\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e154\u003c/strong\u003e, 442–451 (2013).\u003c/li\u003e\n \u003cli\u003eBryant, J. A., Sellars, L. E., Busby, S. J. \u0026amp; Lee, D. J. Chromosome position effects on gene expression in \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eK-12. \u003cem\u003eNucleic Acids Res.\u003c/em\u003e \u003cstrong\u003e42\u003c/strong\u003e, 11383–11392 (2014).\u003c/li\u003e\n \u003cli\u003eMiyake, Y. \u0026amp; Yamamoto, K. Epistatic effect of regulators to the adaptive growth of \u003cem\u003eEscherichia coli\u003c/em\u003e. \u003cem\u003eSci. Rep\u003c/em\u003e. \u003cstrong\u003e10\u003c/strong\u003e, 3661 (2020).\u003c/li\u003e\n \u003cli\u003eKenney, L. J. \u0026amp; Anand, G. S. EnvZ/OmpR Two-component signaling: an archetype system that can function noncanonically. \u003cem\u003eEcoSal Plus\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e, ESP-0001-2019 (2020).\u003c/li\u003e\n \u003cli\u003eHayashi, K. et al. Highly accurate genome sequences of \u003cem\u003eEscherichia coli\u0026nbsp;\u003c/em\u003eK-12 strains MG1655 and W3110. \u003cem\u003eMol. Syst. Biol.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e2\u003c/strong\u003e, 2006.0007 (2006).\u003c/li\u003e\n \u003cli\u003eHelaine, S. et al. Dynamics of intracellular bacterial replication at the single cell level. \u003cem\u003eProc. Natl. Acad. Sci. U. S. A.\u0026nbsp;\u003c/em\u003e\u003cstrong\u003e107\u003c/strong\u003e, 3746–3751 (2010).\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 3 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Escherichia coli, HoSeI method, Multi-cutter plasmid, Gene rewriting; Biosensor","lastPublishedDoi":"10.21203/rs.3.rs-9262883/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9262883/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Precise chromosomal integration of large DNA fragments remains a significant challenge in metabolic engineering of Escherichia coli, largely due to the unpredictable and often low cleavage efficiency of individual single-guide RNAs (sgRNAs). To overcome this issue, we have developed a modular and versatile \"multi-cutters\" platform comprising plasmids designed to express up to four distinct sgRNAs simultaneously from a single vector. Building upon the HoSeI (Homologous Sequence Integration) method, this platform synergizes CRISPR-Cas9-mediated cleavage with λ-Red-mediated repair to enable one-step, scarless editing. Our construction strategy uses a modular assembly approach with specific linkers and restriction sites to enable the rapid and flexible generation of double, triple and quadruple cutters. We demonstrate the utility of this platform by successfully 'rewriting' genes—the one-step replacement of approximately 1 kbp chromosomal regions—by replacing the high-expression ompC and ompF loci with fluorescent reporter genes (mCherry and gfp). The resulting dual-reporter strain, RHA00802, functioned as an effective osmotic biosensor, exhibiting concentration-dependent fluorescence responses to sucrose levels (0–10%) in accordance with the regulation of the EnvZ–OmpR two-component system. This multi-cutter platform and gene rewriting strategy provides a powerful, generalized toolkit for complex genomic design and the rapid construction of functional microbial devices. 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