Fichter, K. M., Setayesh, T. & Malik, P. Strategies for precise gene edits in mammalian cells. Mol. Ther. Nucleic Acids 32, 536–552 (2023).
Google Scholar
Jacinto, F. V., Link, W. & Ferreira, B. I. CRISPR/Cas9-mediated genome editing: from basic research to translational medicine. J. Cell. Mol. Med. 24, 3766–3778 (2020).
Google Scholar
Jang, H.-K., Song, B., Hwang, G.-H. & Bae, S. Current trends in gene recovery mediated by the CRISPR–Cas system. Exp. Mol. Med. 52, 1016–1027 (2020).
Google Scholar
Li, L., Hu, S. & Chen, X. Non-viral delivery systems for CRISPR/Cas9-based genome editing: challenges and opportunities. Biomaterials 171, 207–218 (2018).
Google Scholar
Luther, D. C., Lee, Y. W., Nagaraj, H., Scaletti, F. & Rotello, V. M. Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges. Expert Opin. Drug Deliv. 15, 905–913 (2018).
Google Scholar
Wang, H.-X. et al. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem. Rev. 117, 9874–9906 (2017).
Google Scholar
Yip, B. H. Recent advances in CRISPR/Cas9 delivery strategies. Biomolecules 10, 839 (2020).
Google Scholar
David, R. M. & Doherty, A. T. Viral vectors: the road to reducing genotoxicity. Toxicol. Sci. 155, 315–325 (2017).
Google Scholar
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Google Scholar
Bhattarai-Kline, S. et al. Recording gene expression order in DNA by CRISPR addition of retron barcodes. Nature 608, 217–225 (2022).
Google Scholar
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652 (2021).
Google Scholar
Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002 (2023).
Google Scholar
Ellington, A. J. & Reisch, C. R. Efficient and iterative retron-mediated in vivo recombineering in Escherichia coli. Synth. Biol. 7, ysac007 (2022).
Google Scholar
Farzadfard, F. & Lu, T. K. Genomically encoded analog memory with precise in vivo DNA writing in living cell populations. Science 346, 1256272 (2014).
Google Scholar
Fishman, C. B. et al. Continuous multiplexed phage genome editing using recombitrons. Nat. Biotechnol. 43, 1299–1310 (2025).
Google Scholar
González-Delgado, A., Lopez, S. C., Rojas-Montero, M., Fishman, C. B. & Shipman, S. L. Simultaneous multi-site editing of individual genomes using retron arrays. Nat. Chem. Biol. 20, 1482–1492 (2024).
Google Scholar
Hwang, J., Ye, D.-Y., Jung, G. Y. & Jang, S. Mobile genetic element-based gene editing and genome engineering: recent advances and applications. Biotechnol. Adv. 72, 108343 (2024).
Google Scholar
Jiang, W., et al. High-efficiency retron-mediated single-stranded DNA production in plants. Synth. Biol. 7, ysac025 (2022).
Google Scholar
Kaur, N. & Pati, P. K. Retron library recombineering: next powerful tool for genome editing after CRISPR/Cas. ACS Synth. Biol. 13, 1019–1025 (2024).
Google Scholar
Khan, A. G. et al. An experimental census of retrons for DNA production and genome editing. Nat. Biotechnol. 43, 914–922 (2025).
Google Scholar
Kong, X. et al. Precise genome editing without exogenous donor DNA via retron editing system in human cells. Protein Cell 12, 899–902 (2021).
Google Scholar
Lee, G. and Kim, J. Engineered retrons generate genome-independent protein-binding DNA for cellular control. Preprint at bioRxiv https://doi.org/10.1101/2023.09.27.556556 (2023).
Lim, H. et al. Multiplex generation, tracking, and functional screening of substitution mutants using a CRISPR/retron system. ACS Synth. Biol. 9, 1003–1009 (2020).
Google Scholar
Lin, Q. et al. Prime genome editing in rice and wheat. Nat. Biotechnol. 38, 582–585 (2020).
Google Scholar
Liu, J. et al. Generation of DNAzyme in bacterial cells by a bacterial retron system. ACS Synth. Biol. 13, 300–309 (2024).
Google Scholar
Liu, W. et al. Retron-mediated multiplex genome editing and continuous evolution in Escherichia coli. Nucleic Acids Res. 51, 8293–8307 (2023).
Google Scholar
Lopez, S. C., Crawford, K. D., Lear, S. K., Bhattarai-Kline, S. & Shipman, S. L. Precise genome editing across kingdoms of life using retron-derived DNA. Nat. Chem. Biol. 18, 199–206 (2022).
Google Scholar
Ramirez-Chamorro, L., Boulanger, P. & Rossier, O. Strategies for bacteriophage T5 mutagenesis: expanding the toolbox for phage genome engineering. Front. Microbiol. 12, 667332 (2021).
Google Scholar
Roy, K. R., et al. Dissecting quantitative trait nucleotides by saturation genome editing. Preprint at bioRxiv https://doi.org/10.1101/2024.02.02.577784 (2024).
Schubert, M. G., et al. High-throughput functional variant screens via in vivo production of single-stranded DNA. Proc. Natl Acad. Sci. USA 118, e2018181118 (2021).
Google Scholar
Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557 (2018).
Google Scholar
Simon, A. J., Ellington, A. D. & Finkelstein, I. J. Retrons and their applications in genome engineering. Nucleic Acids Res. 47, 11007–11019 (2019).
Google Scholar
Simon, A. J., Morrow, B. R. & Ellington, A. D. Retroelement-based genome editing and evolution. ACS Synth. Biol. 7, 2600–2611 (2018).
Google Scholar
Tang, S. & Sternberg, S. H. Genome editing with retroelements. Science 382, 370–371 (2023).
Google Scholar
Zhao, B., Chen, S.-A. A., Lee, J. & Fraser, H. B. Bacterial retrons enable precise gene editing in human cells. CRISPR J. 5, 31–39 (2022).
Google Scholar
Azam, A. H. et al. Evasion of antiviral bacterial immunity by phage tRNAs. Nat. Commun. 15, 9586 (2024).
Google Scholar
Bobonis, J. et al. Bacterial retrons encode phage-defending tripartite toxin–antitoxin systems. Nature 609, 144–150 (2022).
Google Scholar
Carabias, A. et al. Retron-eco1 assembles NAD+-hydrolyzing filaments that provide immunity against bacteriophages. Mol. Cell 84, 2185–2202 (2024).
Google Scholar
Gao, L. et al. Diverse enzymatic activities mediate antiviral immunity in prokaryotes. Science 369, 1077–1084 (2020).
Google Scholar
Millman, A. et al. Bacterial retrons function in anti-phage defense. Cell 183, 1551–1561 (2020).
Google Scholar
Palka, C., Fishman, C. B., Bhattarai-Kline, S., Myers, S. A. & Shipman, S. L. Retron reverse transcriptase termination and phage defense are dependent on host RNase H1. Nucleic Acids Res. 50, 3490–3504 (2022).
Google Scholar
Rychlik, I., Sebkova, A., Gregorova, D. & Karpiskova, R. Low-molecular-weight plasmid of Salmonella enterica serovar Enteritidis codes for retron reverse transcriptase and influences phage resistance. J. Bacteriol. 183, 2852–2858 (2001).
Google Scholar
Wang, Y. et al. Cryo-EM structures of Escherichia coli Ec86 retron complexes reveal architecture and defence mechanism. Nat. Microbiol. 7, 1480–1489 (2022).
Google Scholar
Wang, Y., et al. DNA methylation activates retron Ec86 filaments for antiphage defense. Cell Rep. 43, 114857 (2024).
Google Scholar
Hsu, M. Y., Eagle, S. G., Inouye, M. & Inouye, S. Cell-free synthesis of the branched RNA-linked msDNA from retron-Ec67 of Escherichia coli. J. Biol. Chem. 267, 13823–13829 (1992).
Google Scholar
Shimamoto, T., Inouye, M. & Inouye, S. The formation of the 2′,5′-phosphodiester linkage in the cDNA priming reaction by bacterial reverse transcriptase in a cell-free system. J. Biol. Chem. 270, 581–588 (1995).
Google Scholar
Schimmel, J., et al. Modulating mutational outcomes and improving precise gene editing at CRISPR–Cas9-induced breaks by chemical inhibition of end-joining pathways. Cell Rep. 42, 112019 (2023).
Google Scholar
Savic, N. et al. Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair. Elife 7, e33761 (2018).
Google Scholar
Almeida, A. et al. A unified catalog of 204,938 reference genomes from the human gut microbiome. Nat. Biotechnol. 39, 105–114 (2021).
Google Scholar
Pruitt, K. D., Tatusova, T. & Maglott, D. R. NCBI reference sequences (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins. Nucleic Acids Res. 35, D61–D65 (2007).
Google Scholar
Mestre, M. R., González-Delgado, A., Gutiérrez-Rus, L. I., Martínez-Abarca, F. & Toro, N. Systematic prediction of genes functionally associated with bacterial retrons and classification of the encoded tripartite systems. Nucleic Acids Res. 48, 12632–12647 (2020).
Google Scholar
Matreyek, K. A., Stephany, J. J., Chiasson, M. A., Hasle, N. & Fowler, D. M. An improved platform for functional assessment of large protein libraries in mammalian cells. Nucleic Acids Res. 48, e1 (2020).
Google Scholar
Hussmann, J. A. et al. Mapping the genetic landscape of DNA double-strand break repair. Cell 184, 5653–5669 (2021).
Google Scholar
Nambiar, T. S., Baudrier, L., Billon, P. & Ciccia, A. CRISPR-based genome editing through the lens of DNA repair. Mol. Cell 82, 348–388 (2022).
Google Scholar
Potapov, V. et al. Base modifications affecting RNA polymerase and reverse transcriptase fidelity. Nucleic Acids Res. 46, 5753–5763 (2018).
Google Scholar
Yasukawa, K. et al. Next-generation sequencing-based analysis of reverse transcriptase fidelity. Biochem. Biophys. Res. Commun. 492, 147–153 (2017).
Google Scholar
Goeckel, M. E. et al. Modulating CRISPR gene drive activity through nucleocytoplasmic localization of Cas9 in S. cerevisiae. Fungal Biol. Biotechnol. 6, 2 (2019).
Google Scholar
Liu, P. et al. Improved prime editors enable pathogenic allele correction and cancer modelling in adult mice. Nat. Commun. 12, 2121 (2021).
Google Scholar
Luk, K. et al. Optimization of nuclear localization signal composition improves CRISPR–Cas12a editing rates in human primary cells. GEN Biotechnol. 1, 271–284 (2022).
Google Scholar
Maggio, I. et al. Integrating gene delivery and gene-editing technologies by adenoviral vector transfer of optimized CRISPR–Cas9 components. Gene Ther. 27, 209–225 (2020).
Google Scholar
Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).
Google Scholar
Wu, Y. et al. Highly efficient therapeutic gene editing of human hematopoietic stem cells. Nat. Med. 25, 776–783 (2019).
Google Scholar
Inouye, S., Hsu, M.-Y., Xu, A. & Inouye, M. Highly specific recognition of primer RNA structures for 2-OH priming reaction by bacterial reverse transcriptases. J. Biol. Chem. 274, 31236–31244 (1999).
Google Scholar
Tan, J., Zhang, F., Karcher, D. & Bock, R. Engineering of high-precision base editors for site-specific single nucleotide replacement. Nat. Commun. 10, 439 (2019).
Google Scholar
Tanenbaum, M. E., Gilbert, L. A., Qi, L. S., Weissman, J. S. & Vale, R. D. A protein-tagging system for signal amplification in gene expression and fluorescence imaging. Cell 159, 635–646 (2014).
Google Scholar
Hinrichsen, M. et al. A new method for post-translationally labeling proteins in live cells for fluorescence imaging and tracking. Protein Eng., Des. Sel. 30, 771–780 (2017).
Google Scholar
Zhang, L. et al. AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines. Nat. Commun. 12, 3908 (2021).
Google Scholar
Carusillo, A. & Mussolino, C. DNA damage: from threat to treatment. Cells 9, 1665 (2020).
Google Scholar
Fok, J. H. L. et al. AZD7648 is a potent and selective DNA-PK inhibitor that enhances radiation, chemotherapy and olaparib activity. Nat. Commun. 10, 5065 (2019).
Google Scholar
Fu, Y.-W. et al. Dynamics and competition of CRISPR–Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing. Nucleic Acids Res. 49, 969 (2021).
Google Scholar
Riesenberg, S., et al. Simultaneous precise editing of multiple genes in human cells. Nucleic Acids Res. 47, e116 (2019).
Google Scholar
Wimberger, S. et al. Simultaneous inhibition of DNA-PK and Polθ improves integration efficiency and precision of genome editing. Nat. Commun. 14, 4761 (2023).
Google Scholar
Iwai, K., et al. Molecular mechanism and potential target indication of TAK-931, a novel CDC7-selective inhibitor. Sci. Adv. 5, eaav3660 (2019).
Google Scholar
Carusillo, A. et al. A novel Cas9 fusion protein promotes targeted genome editing with reduced mutational burden in primary human cells. Nucleic Acids Res. 51, 4660–4673 (2023).
Google Scholar
Jayavaradhan, R. et al. CRISPR–Cas9 fusion to dominant-negative 53BP1 enhances HDR and inhibits NHEJ specifically at Cas9 target sites. Nat. Commun. 10, 2866 (2019).
Google Scholar
Reint, G. et al. Rapid genome editing by CRISPR–Cas9–POLD3 fusion. Elife 10, e75415 (2021).
Google Scholar
Gutschner, T., Haemmerle, M., Genovese, G., Draetta, G. F. & Chin, L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep. 14, 1555–1566 (2016).
Google Scholar
Arslan, S., Khafizov, R., Thomas, C. D., Chemla, Y. R. & Ha, T. Engineering of a superhelicase through conformational control. Science 348, 344–347 (2015).
Google Scholar
Morrison, C. et al. The essential functions of human RAD51 are independent of ATP hydrolysis. Mol. Cell. Biol. 19, 6891–6897 (1999).
Google Scholar
Barondeau, D. P., Putnam, C. D., Kassmann, C. J., Tainer, J. A. & Getzoff, E. D. Mechanism and energetics of green fluorescent protein chromophore synthesis revealed by trapped intermediate structures. Proc. Natl Acad. Sci. USA 100, 12111–12116 (2003).
Google Scholar
Cabantous, S., Terwilliger, T. C. & Waldo, G. S. Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein. Nat. Biotechnol. 23, 102–107 (2005).
Google Scholar
Kamiyama, D. et al. Versatile protein tagging in cells with split fluorescent protein. Nat. Commun. 7, 11046 (2016).
Google Scholar
Pinaud, F. & Dahan, M. Targeting and imaging single biomolecules in living cells by complementation-activated light microscopy with split-fluorescent proteins. Proc. Natl Acad. Sci. USA 108, E201–E210 (2011).
Google Scholar
Feng, J. et al. Generation and characterization of tamoxifen-inducible Pax9-CreER knock-in mice using CRISPR/Cas9. Genesis 54, 490–496 (2016).
Google Scholar
Kehler, J. et al. RNA-generated and gene-edited induced pluripotent stem cells for disease modeling and therapy. J. Cell. Physiol. 232, 1262–1269 (2017).
Google Scholar
Stadelmann, C. et al. mRNA-mediated delivery of gene editing tools to human primary muscle stem cells. Mol. Ther. Nucleic Acids 28, 47–57 (2022).
Google Scholar
Yin, H. et al. Structure-guided chemical modification of guide RNA enables potent non-viral in vivo genome editing. Nat. Biotechnol. 35, 1179–1187 (2017).
Google Scholar
Konjikusic, M. J. et al. Mutations in kinesin family member 6 reveal specific role in ependymal cell ciliogenesis and human neurological development. PLoS Genet. 14, e1007817 (2018).
Google Scholar
Aird, E. J., Lovendahl, K. N., St. Martin, A., Harris, R. S. & Gordon, W. R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template. Commun. Biol. 1, 1–6 (2018).
Google Scholar
Carlson-Stevermer, J. et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 8, 1711 (2017).
Google Scholar
Rees, H. A. & Liu, D. R. Base editing: precision chemistry on the genome and transcriptome of living cells. Nat. Rev. Genet. 19, 770–788 (2018).
Google Scholar
Zhao, Z., Shang, P., Mohanraju, P. & Geijsen, N. Prime editing: advances and therapeutic applications. Trends Biotechnol. 41, 1000–1012 (2023).
Google Scholar
Bothmer, A. et al. Characterization of the interplay between DNA repair and CRISPR/Cas9-induced DNA lesions at an endogenous locus. Nat. Commun. 8, 13905 (2017).
Google Scholar
Engler, C. et al. A golden gate modular cloning toolbox for plants. ACS Synth. Biol. 3, 839–843 (2014).
Google Scholar
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023).
Google Scholar
Gallagher, D. N. & Haber, J. E. Single-strand template repair: key insights to increase the efficiency of gene editing. Curr. Genet. 67, 747–753 (2021).
Google Scholar
Gallagher, D. N., et al. A RAD51-independent pathway promotes single-strand template repair in gene editing. PLoS Genet. 16, e1008689 (2020).
Google Scholar
Richardson, C. D. et al. CRISPR–Cas9 genome editing in human cells occurs via the fanconi anemia pathway. Nat. Genet. 50, 1132–1139 (2018).
Google Scholar
Okamoto, S., Amaishi, Y., Maki, I., Enoki, T. & Mineno, J. Highly efficient genome editing for single-base substitutions using optimized ssODNs with Cas9-RNPs. Sci. Rep. 9, 4811 (2019).
Google Scholar
Schubert, M. S. et al. Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair. Sci. Rep. 11, 19482 (2021).
Google Scholar
Paulsen, B. S. et al. Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing. Nat. Biomed. Eng. 1, 878–888 (2017).
Google Scholar
Wyatt, D. W. et al. Essential roles for polymerase θ-mediated end joining in the repair of chromosome breaks. Mol. Cell 63, 662–673 (2016).
Google Scholar
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR–Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Google Scholar
Eddy, S. R. Accelerated profile HMM searches. PLoS Comput. Biol. 7, e1002195 (2011).
Google Scholar
Finn, R. D., Clements, J. & Eddy, S. R. HMMER web server: interactive sequence similarity searching. Nucleic Acids Res. 39, W29–W37 (2011).
Google Scholar
Yao, Z., Weinberg, Z. & Ruzzo, W. L. CMfinder—a covariance model based RNA motif finding algorithm. Bioinformatics 22, 445–452 (2006).
Google Scholar
Lorenz, R. et al. ViennaRNA Package 2.0. Algorithms Mol. Biol. 6, 26 (2011).
Google Scholar
Fu, L., Niu, B., Zhu, Z., Wu, S. & Li, W. CD-HIT: accelerated for clustering the next-generation sequencing data. Bioinformatics 28, 3150–3152 (2012).
Google Scholar
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
Google Scholar
Nguyen, L.-T., Schmidt, H. A., Von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).
Google Scholar
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Google Scholar
Meeker, N. D., Hutchinson, S. A., Ho, L. & Trede, N. S. Method for isolation of PCR-ready genomic DNA from zebrafish tissues. Biotechniques 43, 610–614 (2007).
Google Scholar
