Unveiling the hidden window of prime editing

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Unveiling the hidden window of prime editing View ORCID Profile Pei-Ru Chen , View ORCID Profile Ying Wei , View ORCID Profile Xian-Zheng Yuan , View ORCID Profile Shu-Guang Wang , View ORCID Profile Peng-Fei Xia doi: https://doi.org/10.1101/2025.05.20.655067 Pei-Ru Chen 1 School of Environmental Science and Engineering, Shandong University , Qingdao 266237, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pei-Ru Chen Ying Wei 1 School of Environmental Science and Engineering, Shandong University , Qingdao 266237, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Ying Wei Xian-Zheng Yuan 1 School of Environmental Science and Engineering, Shandong University , Qingdao 266237, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Xian-Zheng Yuan Shu-Guang Wang 1 School of Environmental Science and Engineering, Shandong University , Qingdao 266237, China 2 Sino-French Research Institute for Ecology and Environment, Shandong University , Qingdao 266237, China 3 Weihai Research Institute of Industrial Technology, Shandong University , Weihai 264209, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Shu-Guang Wang Peng-Fei Xia 1 School of Environmental Science and Engineering, Shandong University , Qingdao 266237, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Peng-Fei Xia For correspondence: pfxia{at}sdu.edu.cn Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Prime editing enables versatile and precise genomic modifications, following a fundamental principle that the editing extends from the nick site towards the 3’ direction of the cleaved DNA strand. Here, we report the unknown competency of prime editing on the opposite 5’ direction of the nicked DNA strand through a hidden reverse transcription pathway, where a designed edit can be integrated upstream of the nick. Prime editing utilizes CRISPR-Cas system and reverse transcription to enable versatile genome editing with minimal unintended perturbations, opening new avenues for next-generation medicine and agriculture 1 - 3 . It was invented by leveraging a fused Cas9 nickase (nCas9) and reverse transcriptase (RT), and a prime editing guide RNA (pegRNA) with primer binding site (PBS) and reverse transcription template (RTT) adding to a single guide RNA 4 . Specifically, nCas9 cleaves the single strand DNA within the formed R-Loop, generating a nick, where the exposed 3’-hydroxyl group primes the reverse transcription using RTT as a substrate ( Fig. 1a ) . Through subsequent biological processing, the designed edits in RTT are integrated into the genome 4 . Despite advancements in effectors 5 - 8 and pegRNAs 9 - 11 , all prime editing systems, without a single exception, follow this fundamental principle, thereby synergizing an editing capacity extending from the nick towards the 3’ direction of the cleaved DNA strand ( Fig. 1a ) . Only, we discovered that the well-received editing pathway is not inclusive. Here, we report the unknown competency of prime editing in the opposite 5’ direction through a hidden pathway for reverse transcription, where an intended edit can be integrated upstream of the nick. Download figure Open in new tab Fig 1. Prime editing on the 5’ direction of the nick. (a) Design of prime editing relies on reverse transcription primed by the exposed 3’-hydroxyl-group generated by DNA cleavage. The reverse transcription extends from locus +1 towards the 3’ direction to synthesize a new DNA strand containing intended edits using RTT as the substrate. The mechanism synergizes an editing capacity downstream of the nick, making editing on the opposite 5’ direction impractical. The first base downstream of the nick as locus +1. (b) Experimental scheme to evaluate the editing capacity using customized PE2 targeting a plasmid-carrying gfp and the chromosomal pta in E. coli . (c) Designs of pegRNAs targeting gfp and (e) pta with single-nucleotide substitution or continuous substitutions in the 5’ direction of the nick. Edits on RTT are designed as an indication of editing efficacy. (d) Representative sequencing results upstream of the nick with gfp as the target. (f) Sequencing demonstration of editing on the chromosome. Successful edits are highlighted at the designed loci. (g) Prime editing efficiencies for single-nucleotide substitution and (h) continuous substitutions with a 13-nt PBS. (i,j) Prime editing efficiency with a 15-nt PBS. Three biological independent experiments were performed with error bars indicating standard deviations. Statistical significance is determined by t -test (*, P <0.05; **, P <0.01; ***, P <0.001). To evaluate the uncovered capacity, we customized the PE2 4 for Escherichia coli , and we targeted a plasmid-carried gfp as the gene of interest (GOI) to avoid efficiency-misled incapability ( Fig. 1b ) . First, we designed pegRNAs with single nucleotide substitution on the PBS with an edit on RTT serving as a positive control indicating the efficacy of prime editing ( Fig. 1c ) . With the first position downstream of the nick as locus +1 ( Fig. 1a ) , the editing loci ranged from -1 to -7, leaving at least 6 nt in a 13-nt PBS as the binding site 4 . We observed successful editing from loci -1 to -6, while locus -7 could not be modified ( Fig. 1d , Extended Data Fig.1 ) . Next, we designed pegRNAs containing edits with continuous substitutions ranging from 2 to 7 nt ( Fig. 1c ) , and a maximal 6-nt substitution (-6 to -1 GTTCAC) was achieved ( Fig. 1d , Extended Data Fig.1 ) . These results indicated prime editing could reach out locus -6 with a 13-nt PBS. Due to sequence preference 4 , the success of 5-nt editing was observed with varied editing sequences on locus -5 (-5 to -1 GTCAC and CTCAC) instead of the original design (-5 to -1 TTCAC) ( Extended Data Fig.1 ) . Taking locus -3 as an example, we found that the editing upstream of the nick could be achieved alone without accompanied edits in RTT, and all the three types of editing, substitution, insertion and deletion, were possible ( Figs. 1c,1d ) . In addition, we targeted the pta gene in the chromosome of E. coli ( Fig. 1b ), and, as designed, successful editing was identified on the previously unknown direction ( Figs. 1e,1f ) . Download figure Open in new tab Extended Data Fig 1. Sanger sequencing results. (a) Sequencing results of single-nucleotide substitution and (b) continuous nucleotide substitutions with pegRNAs containing 13-nt PBS. Successful edits are highlighted at the designed loci. For single nucleotide editing, we found the efficiency decreased with the increased distance from the nick until locus -6 ( Fig. 1g ) . The efficiency of continuous substitutions also decreased with the extend length ( Fig. 1h ) . These data suggested a minimal requirement of binding site and decreased binding sites reducing the editing efficiency, which aligned with previous knowledge of PBS 4 , 12 . As such, we elongated the PBS to 15 nt ( Extended Data Fig. 2 ). While the single nucleotide substitution at locus -7 failed ( Fig. 1i ) , we successfully obtained the -7 to -1 AGTTCAC substitutions and the previously unsuccessful -5 to -1 TTCAC substitutions ( Fig. 1j , Extended Data Fig. 2 ) . Taking together, we demonstrated the capacity of prime editing on the opposite 5’ direction of nick. The editing could be obtained in an editing window of -1 to -6 with a 13-nt PBS, while it is possible to expand the window with prolonged binding sites. Download figure Open in new tab Extended Data Fig 2. Prime editing with pegRNAs with 15-nt PBS. (a) Design of pegRNAs with single-nucleotide substitution or continuous substitutions with 15-nt PBS. (b) Sanger sequencing results of prime editing. Successful edits are highlighted at the designed loci. The editing upstream of the nick is theoretically impossible through the received reverse transcription pathway (rRTP). We conceive a hidden reverse transcription pathway (hRTP) that initiates at the designed edit that mismatches the DNA strand ( Fig. 2a ) . While part of the PBS functionally serves as the RTT in hRTP, we retained the terminology and introduced reverse transcription start site (RTS) to illustrate the mechanism. When edits on both sides were designed, we observed that the editing upstream of the nick always accompanied the edits on the 3’ direction, while no standalone editing has been identified, suggesting a single reverse-transcribed DNA orchestrating the editing. This can be immediately justified by the 3-to 7-nt continuous substitutions upstream of the nick, where similar efficiencies were determined on both sides ( Figs. 1h,1j ) . Lacking an exposed 3’-hydroxyl group before RTS, the new DNA strand can be synthesized through hRTP initiated with non-3’-hydroxyl-group-mediated priming, such as random small DNAs, which has long been proven as an efficient machinery for reverse transcription 13 . The resulting 3’ flap would go through a post reverse transcription process mimicking the subsequent flap cleavage and DNA ligation 4 , forming the new DNA strand with intended editing starting from RTS rather than the nick ( Fig. 2a ) . The distinct editing efficiencies of single substitutions on both sides of the nick can be explained by the competition between hRTP and rRTP ( Fig. 2a ) . Different from continuous substitutions, a single mismatch in the middle of the PBS could barely eliminate rRTP, as rest of the nucleotides still bind to the PBS and a 3’-hydroxyl group is exposed to initiate rRTP. As each editing is mediated by a single reverse-transcribed DNA strand, the outcome is determined by the pathway. Only if the editing goes through hRTP, the intended edits can be integrated ( Fig. 2a ) . The competition between hRTP and rRTP could also elucidate the unique 2-nt editing profile of -1 to -2 substitutions ( Fig. 1h ) . Download figure Open in new tab Fig 2. Pathways for prime editing. (a) hRTP enables editing upstream of the nick. The reverse transcription initiates at the RTS rather than the nick through non-3’-hydroxyl-group-mediated priming. The product goes through post-reverse transcription process to form a DNA strand containing the designed edits, which is eventually integrated after subsequent biological processes. The pathway determines the outcome of prime editing, where rRTP competes with hRTP. (b) Prime editing efficiencies in MMR-deficient strains. Error bars represent the standard deviation from three independent biological replicates. Statistical significance is determined by t - test (**, P <0.01; ***, P <0.001). hRTP, hidden reverse transcription pathway; rRTP, received reverse transcription pathway; RTS, reverse transcription start site. The efficiency could not directly link to the competency of hRTP as part of the edits were removed by mismatch repair (MMR) 14 , 15 . The disruption of MMR ( Extended Data Fig. 3 ) exerted limited effects on single substitutions upstream of the nick, maintaining an efficiency around 15.18%, while the edits downstream of the nick approaching 99.31%, implying an already saturated overall efficiency ( Fig. 2b ) . These differences indicated the competing capacities of the two pathways from RTS and the nick. To the contrary, the 3-nt continuous substitutions were significantly enhanced in mutS and mutLS deficient strains, reaching a maximum of 95.83%, suggesting 75.22% of the edits were removed by MMR in the wild-type strain (20.61%) ( Fig. 2b ) . Therefore, rRTP, when it can be initiated, overwhelms hRTP, repressing the editing efficiency upstream of the nick, and the pathway-competition-led low efficiency could not be rescued in a later process. Indeed, it has now become difficult to determine whether the rRTP primed by the exposed 3’-hydroxyl group stands alone or represents a hybridization of different pathways, as RTS coincides the nick. Download figure Open in new tab Extended Data Fig 3. Disruption of MMR. (a) Design of the base editing plasmid. The editing plasmid contains an inducible lacI -P trc system driving the fused dCas9, PmCDA1, ugi and LVA tag, and gRNA cassettes led by P J23119 promoter targeting mutL and mutS . The plasmid includes a temperature-sensitive origin of replication oriR101 , and a bla gene as the selection marker. (b) Sequencing results of edited mutL in PR20), (c) mutS in PR21, and (d) both genes in PR22. Successful edits are highlighted at the designed loci, and the introduced STOP codon are also highlighted. To conclude, we unveil the unknown competency of prime editing in the 5’ direction of the nicked DNA strand. This is impossible through the previously undoubtable mechanism, but enabled by a hidden pathway of reverse transcription initiated by non-3’-hydroxyl-group-mediated priming from the RTS rather than a nick. While the exact mechanism remains to be elucidated, we envision a revolutionary change in understanding and creating prime editing systems. Methods Strains and media E. coli DH5α (Takara) was used for molecular cloning in general. E. coli MG1655 was employed as the wild-type strain, and it was transformed with a gfp -carrying plasmid, generating PR19, for evaluating the unknown competency of prime editing. To evaluate DNA mismatch repair (MMR) 16 , 17 , we constructed MMR-deficient strains PR20 ( mutL Gln4*), PR21 ( mutS Gln138*), and PR22 ( mutL Gln4* and mutS Gln138*). The MMR-mutant strains were subsequently transformed with the gfp -carrying plasmid for editing experiments, generating PR45, PR46, and PR47, respectively. These were further transformed with the prime editing plasmids for gene editing. All strains used and generated in this study are listed in Table S1. E. coli strains were grown in Luria-Bertani (LB) medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) and selected in LB solid media (with 1.5% agar). Carbenicillin (100 µg/mL) and gentamicin (20 µg/mL) were supplemented for plasmid selection and maintenance. Isopropyl-β-D-thiogalactoside (IPTG) was added to a final concentration of 1 mM to induce prime editing and base editing. Chemically competent cells were prepared using a CaCl 2 protocol 18 . Plasmid construction General DNA manipulation and molecular cloning were conducted as follows. PCR amplification of DNA fragments was performed using PrimeSTAR ® Max DNA Polymerase (Takara), and plasmid assembly was achieved via the In-Fusion ® Snap Assembly Master Mix (Takara). Plasmids were extracted using the QIAprep Spin Miniprep Kit (QIAGEN) and DNA purification was carried out using the QIAquick PCR Purification Kit (QIAGEN). Gel electrophoresis and Sanger sequencing were employed for plasmid validation. DNA concentration was quantified by NanoDrop One (Thermo Scientific). To investigate the capacity of prime editing, we constructed the target plasmid pGFP using pBR322 19 as a backbone with the pBBR1MCS-5 oriV and pBBR1MCS-5 rep 20 and bla as the selection marker. The gfp gene from pAM4787 21 was incorporated as the gene of interest (GOI). Next, we customized PE2 for E. coli , employing a previously established prime editing system, pPE.S, containing the engineered Cas9 nickase (nCas9, H840A) from Streptococcus pyogenes fused to an E. coli codon-optimized M-MLV reverse transcriptase (RT) under the control of a lacI -P trc inducible system 22 . The oriR101 in pPE.S was replaced with the p15A ori for better performance, generating the working plasmid template pRC. The pegRNA was commercially synthesized on a pUC57 plasmid driven by a constitutive promoter P J23119 . Then, the pegRNA cassettes were amplified and incorporated into pRC to generate the pRC12 - pRC38 serial editing plasmids (Tables S2 and S3) . To investigate the role of MMR in prime editing, we employed the established E. coli cytosine base editing system, pBeCas9 23 , to inactivate MMR genes. The system contains dCas9, PmCDA1, ugi and an LVA tag under the control of a lacI -P trc inducible system, along with a temperature-sensitive origin of replication oriR101 and bla as the selection marker. The P J23119 -driven gRNA cassettes were generated via inverse PCR and were incorporated into pBeCas9 via gene assembly, generating base editing working plasmids. All gRNA sequences are provided in Table S2 and plasmids are provided in Table S3 . All primers used in this study are listed in Table S4. Mutating MMR with base editing We employed base editing to disrupt MMR by introducing premature STOP codons into mutS and mutL (Table S5) . The Gln4 in mutL and Gln138 in mutS (both encoded by CAG) were converted to STOP codons (TAG) with specifically designed gRNA-mutL and gRNA-mutS (Table S2) . To generate the mutated strains, the wild-type MG1655 were edited with pBeCas9-mutL and pBeCas9-mutS, respectively, to generate PR20 and PR21. PR22 was constructed by editing PR20 with pBeCas9-mutS (Table S3). All base editing was transformed with 25 ng of plasmid DNA via heat shock. Then, the cells were recovered in LB at 30 ºC for 1 h, followed by induction with 1 mM IPTG for 4 h at 30 ºC. The edited strains were selected on solid media with appropriate antibiotics. Successful editing was confirmed by Sanger sequencing. After curing the base editing plasmids, PR20, PR21 and PR22 were transformed with the pGFP (Table S3) for prime editing evaluation. Design of pegRNAs We designed pegRNAs targeting gfp on pGFP and pta on the chromosome to evaluate the capacity of prime editing. The pegRNAs used a 13-nt PBS and 13-nt RTT unless otherwise specified. Targeting the plasmid-carrying gfp , single nucleotide substitutions from loci -1 to -7 (pegRNA02 - pegRNA08) and continuous substitutions ranging from 2 to 7 nt (pegRNA09 - pegRNA14, pegRNA21 and pegRNA22) were designed with an intended editing at locus +5 in RTT to indicate the efficacy of prime editing. pegRNA01 was designed for +5 substitution. Since the initial design for 5-nt continuous substitutions (pegRNA12, -5 to -1 TTCAC) failed, we designed two alternative pegRNAs (pegRNA21, -5 to -1 GTCAC; pegRNA22, -5 to -1 CTCAC) for 5-nt continuous substitutions. Next, we designed pegRNA23, pegRNA24 and pegRNA25 for gene substitution, insertion, and deletion at locus -3 without including edits in RTT. Subsequently, we designed pegRNAs with a 15-nt PBS, including pegRNA18 and pegRNA19 for single substitutions at loci -3 and -7, and pegRNA17, pegRNA15, and pegRNA20 for continuous substitutions of 3 nt, 5 nt and 7 nt upstream of the nick. Targeting the chromosomal pta , we designed pegRNAs to make +6 substitution (pegRNA26), -3 substitution (pegRNA27) and -3 to -1 continuous substitutions (pegRNA28). PegRNA27 and pegRNA28 also contain edits at locus +6. All pegRNAs were commercially synthesized with their sequences provided in Table S2. Prime editing The wild-type E. coli MG1655 and pGFP-containing strain PR19 (Table S1) were employed as the target strains. For plasmid editing, we transformed PR19 with prime editing plasmids (pRC12 - pRC35) (Table S3), generating correspondingly edited strains PR27 - PR44 (Table S1) . For chromosome editing, we directly transformed the wild-type E. coli MG1655 with the editing plasmids (pRC36 - pRC38) targeting pta , creating PR23 - PR25. All prime editing was transformed with 25 ng of individual plasmid DNA via heat shock. Cells were recovered in LB medium for 1 h (with 100 µg mL -1 carbenicillin for plasmid editing). The cells were then induced for 24 h in fresh LB medium containing 1 mM IPTG and antibiotics (100 µg mL -1 carbenicillin and 20 µg mL -1 gentamicin for plasmid editing; 20 µg mL -1 gentamicin for genome editing). When necessary, we extended the induction time for chromosomal prime editing. Successful editing was confirmed by Sanger sequencing of the target genomic region. Sanger sequencing The targeted DNA sequences of randomly selected colonies were amplified via PCR. The PCR products were Sanger sequenced to check the edits. The raw sequencing results were aligned to their respective reference sequences in SnapGene 8.0.3. Chromosomal loci pta (ECK2291), mutS (ECK2728), and mutL (ECK4166), were aligned to the E. coli K-12 MG1655 reference genome (the National Center for Biotechnology Information accession number NC_000913 ), while the gfp sequence was aligned to correlated sequence on plasmid pAM4787 (Addgene #120088, generously provided by Dr. Susan Golden’s laboratory). All reference sequences are provided in Table S5. Analysis of editing efficiency After determining the edits through Sanger sequencing, we observed some of the colonies containing mixed sequence signals. This was in agreement with previous reports on prime editing and base editing, while the editing could be purified through one-step segregation. To calculate the efficiency, both pure and mixed sequencing signals were counted as edited. The efficiency was calculated as the percentage of successfully edited colonies among all sequencing colonies (1). Plasmid curing After gene editing, plasmids were cured by culturing the edited strains in antibiotic-free LB medium at 37 ºC for 24 h (supplemented with carbenicillin for maintaining pGFP when necessary), followed by streaking on LB agar plates to obtain individual colonies. Successful plasmid curing was confirmed by determining the plasmid-specific sequences and antibiotic sensitivity to gentamicin. Statistical analysis Three biological independent replications were performed for all experiments, and the data are presented as mean ± standard deviation. Significance was determined by two- tailed unpaired t -test (*, P <0.05; **, P <0.01; ***, P <0.001). Conflict of Interests The authors declare no conflict of interest. Acknowledgments This work was supported by the National Natural Science Foundation of China (22278246 and 22378233), the Department of Science and Technology of Shandong Province (2022HWYQ-017), the Qilu Young Scholar Program of Shandong University (to P.-F.X.), and the Taishan Scholars Project of Shandong Province (NO. tstp20230604). Funder Information Declared National Natural Science Foundation of China, https://ror.org/01h0zpd94 , 22278246 , 22378233 Department of Science and Technology of Shandong Province, https://ror.org/01b9fvd84 , 2022HWYQ-017 Shandong University, https://ror.org/0207yh398 , Qilu Young Scholar Program Taishan Scholars Project of Shandong Province , NO. tstp20230604 References 1. ↵ Chen , P.J. & Liu , D.R. Prime editing for precise and highly versatile genome manipulation . Nat Rev Genet 24 , 161 – 177 ( 2023 ). OpenUrl CrossRef PubMed 2. Li , B. , Sun , C. , Li , J. & Gao , C. Targeted genome-modification tools and their advanced applications in crop breeding . Nat Rev Genet 25 , 603 – 622 ( 2024 ). OpenUrl CrossRef PubMed 3. ↵ Villiger , L. et al. CRISPR technologies for genome, epigenome and transcriptome editing . Nat Rev Mol Cell Biol 25 , 464 – 487 ( 2024 ). OpenUrl CrossRef PubMed 4. ↵ Anzalone , A.V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA . Nature 576 , 149 – 157 ( 2019 ). OpenUrl CrossRef PubMed 5. ↵ Liu , B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates . Nat Biotechnol 42 , 1039 – 1045 ( 2024 ). OpenUrl CrossRef PubMed 6. Liu , P. et al. Increasing intracellular dNTP levels improves prime editing efficiency . Nat Biotechnol ( 2024 ). 7. Yan , J. et al. Improving prime editing with an endogenous small RNA-binding protein . Nature 628 , 639 – 647 ( 2024 ). OpenUrl CrossRef PubMed 8. ↵ Truong , D.J. et al. Exonuclease-enhanced prime editors . Nat Methods 21 , 455 – 464 ( 2024 ). OpenUrl CrossRef PubMed 9. ↵ Nelson , J.W. et al. Engineered pegRNAs improve prime editing efficiency . Nat Biotechnol 40 , 402 – 410 ( 2022 ). OpenUrl CrossRef PubMed 10. Fei , J. et al. Mismatch prime editing gRNA increased efficiency and reduced indels . Nat Commun 16 , 139 ( 2025 ). OpenUrl CrossRef PubMed 11. ↵ Xiong , Y. et al. EXPERT expands prime editing efficiency and range of large fragment edits . Nat Commun 16 , 1592 ( 2025 ). OpenUrl CrossRef PubMed 12. ↵ Mathis , N. et al. Predicting prime editing efficiency and product purity by deep learning . Nat Biotechnol 41 , 1151 – 1159 ( 2023 ). OpenUrl CrossRef PubMed 13. ↵ Gubler , U. & Hoffman , B.J. A simple and very efficient method for generating cDNA libraries . Gene 25 , 263 – 269 ( 1983 ). OpenUrl CrossRef PubMed Web of Science 14. ↵ Chen , P.J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes . Cell 184 , 5635 – 5652 ( 2021 ). OpenUrl CrossRef PubMed 15. ↵ Ferreira da Silva , J. et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair . Nat Commun 13 , 760 ( 2022 ). OpenUrl PubMed References 16. ↵ Lu , A.L. , Clark , S. & Modrich , P. Methyl-directed repair of DNA base-pair mismatches in vitro . Proc Natl Acad Sci U S A 80 , 4639 – 4643 ( 1983 ). OpenUrl Abstract / FREE Full Text 17. ↵ Chen , P.J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes . Cell 184 , 5635 – 5652 e5629 ( 2021 ). OpenUrl CrossRef PubMed 18. ↵ Cohen , S.N. , Chang , A.C. & Hsu , L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA . Proc Natl Acad Sci U S A 69 , 2110 – 2114 ( 1972 ). OpenUrl Abstract / FREE Full Text 19. ↵ Bolivar , F. et al. Construction and characterization of new cloning vehicles. II. A multipurpose cloning system . Gene 2 , 95 – 113 ( 1977 ). OpenUrl CrossRef PubMed Web of Science 20. ↵ Kovach , M.E. et al. Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes . Gene 166 , 175 – 176 ( 1995 ). OpenUrl CrossRef PubMed Web of Science 21. ↵ Chen , Y. et al. Self-replicating shuttle vectors based on pANS, a small endogenous plasmid of the unicellular cyanobacterium Synechococcus elongatus PCC 7942 . Microbiology (Reading) 162 , 2029 – 2041 ( 2016 ). OpenUrl CrossRef PubMed 22. ↵ Chen , P.R. et al. Precision engineering of the probiotic Escherichia coli Nissle 1917 with prime editing . Appl Environ Microbiol 91 , e0003125 ( 2025 ). OpenUrl CrossRef PubMed 23. ↵ Li , X. , Wei , Y. , Wang , S.Y. , Wang , S.G. & Xia , P.F. One-for-all gene inactivation via PAM-independent base editing in bacteria . J Biol Chem 301 , 108113 ( 2025 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted May 21, 2025. 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Share Unveiling the hidden window of prime editing Pei-Ru Chen , Ying Wei , Xian-Zheng Yuan , Shu-Guang Wang , Peng-Fei Xia bioRxiv 2025.05.20.655067; doi: https://doi.org/10.1101/2025.05.20.655067 Share This Article: Copy Citation Tools Unveiling the hidden window of prime editing Pei-Ru Chen , Ying Wei , Xian-Zheng Yuan , Shu-Guang Wang , Peng-Fei Xia bioRxiv 2025.05.20.655067; doi: https://doi.org/10.1101/2025.05.20.655067 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Synthetic Biology Subject Areas All Articles Animal Behavior and Cognition (7642) Biochemistry (17715) Bioengineering (13907) Bioinformatics (42003) Biophysics (21470) Cancer Biology (18624) Cell Biology (25533) Clinical Trials (138) Developmental Biology (13390) Ecology (19935) Epidemiology (2067) Evolutionary Biology (24356) Genetics (15617) Genomics (22529) Immunology (17753) Microbiology (40432) Molecular Biology (17200) Neuroscience (88681) Paleontology (667) Pathology (2840) Pharmacology and Toxicology (4828) Physiology (7653) Plant Biology (15161) Scientific Communication and Education (2046) Synthetic Biology (4304) Systems Biology (9826) Zoology (2271)