Doudna, J. A. The promise and challenge of therapeutic genome editing. Nature 578, 229–236 (2020).
Google Scholar
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Google Scholar
van Overbeek, M. et al. DNA repair profiling reveals nonrandom outcomes at Cas9-mediated breaks. Mol. Cell 63, 633–646 (2016).
Google Scholar
Shen, M. W. et al. Predictable and precise template-free CRISPR editing of pathogenic variants. Nature 563, 646–651 (2018).
Google Scholar
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765–771 (2018).
Google Scholar
Leibowitz, M. L. et al. Chromothripsis as an on-target consequence of CRISPR–Cas9 genome editing. Nat. Genet. 53, 895–905 (2021).
Google Scholar
Kosicki, M. et al. Cas9-induced large deletions and small indels are controlled in a convergent fashion. Nat. Commun. 13, 3422 (2022).
Google Scholar
Alanis-Lobato, G. et al. Frequent loss of heterozygosity in CRISPR–Cas9-edited early human embryos. Proc. Natl Acad. Sci. USA 118, e2004832117 (2021).
Google Scholar
Enache, O. M. et al. Cas9 activates the p53 pathway and selects for p53-inactivating mutations. Nat. Genet. 52, 662–668 (2020).
Google Scholar
Haapaniemi, E., Botla, S., Persson, J., Schmierer, B. & Taipale, J. CRISPR–Cas9 genome editing induces a p53-mediated DNA damage response. Nat. Med. 24, 927–930 (2018).
Google Scholar
Ihry, R. J. et al. p53 inhibits CRISPR–Cas9 engineering in human pluripotent stem cells. Nat. Med. 24, 939–946 (2018).
Google Scholar
Cullot, G. et al. CRISPR–Cas9 genome editing induces megabase-scale chromosomal truncations. Nat. Commun. 10, 1136 (2019).
Google Scholar
Cullot, G. et al. Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR–Cas9. Nat. Commun. 14, 4072 (2023).
Google Scholar
Boutin, J. et al. CRISPR–Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells. Nat. Commun. 12, 4922 (2021).
Google Scholar
Tsai, H.-H. et al. Whole genomic analysis reveals atypical non-homologous off-target large structural variants induced by CRISPR–Cas9-mediated genome editing. Nat. Commun. 14, 5183 (2023).
Google Scholar
Hustedt, N. & Durocher, D. The control of DNA repair by the cell cycle. Nat. Cell Biol. 19, 1–9 (2017).
Google Scholar
Yeh, C. D., Richardson, C. D. & Corn, J. E. Advances in genome editing through control of DNA repair pathways. Nat. Cell Biol. 21, 1468–1478 (2019).
Google Scholar
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Google Scholar
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (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
Grünewald, J. et al. Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437 (2019).
Google Scholar
Zuo, E. et al. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292 (2019).
Google Scholar
Park, S. & Beal, P. A. Off-target editing by CRISPR-guided DNA base editors. Biochemistry 58, 3727–3734 (2019).
Google Scholar
Huang, T. P., Newby, G. A. & Liu, D. R. Precision genome editing using cytosine and adenine base editors in mammalian cells. Nat. Protoc. 16, 1089–1128 (2021).
Google Scholar
Porto, E. M., Komor, A. C., Slaymaker, I. M. & Yeo, G. W. Base editing: advances and therapeutic opportunities. Nat. Rev. Drug Discovery 19, 839–859 (2020).
Google Scholar
Halperin, S. O. et al. CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window. Nature 560, 248–252 (2018).
Google Scholar
Tou, C. J., Schaffer, D. V. & Dueber, J. E. Targeted diversification in the S. cerevisiae genome with CRISPR-guided DNA polymerase I. ACS Synth. Biol. 9, 1911–1916 (2020).
Google Scholar
Long, M. et al. Directed evolution of ornithine cyclodeaminase using an EvolvR-based growth-coupling strategy for efficient biosynthesis of l-proline. ACS Synth. Biol. 9, 1855–1863 (2020).
Google Scholar
Gossing, M. et al. Multiplexed guide RNA expression leads to increased mutation frequency in targeted window using a CRISPR-guided error-prone DNA polymerase in Saccharomyces cerevisiae. ACS Synth. Biol. 12, 2271–2277 (2023).
Google Scholar
Nakade, S. et al. Frame editors for precise, template-free frameshifting. Preprint at https://doi.org/10.1101/2022.12.05.518807 (2022).
Yang, Q. et al. Phage DNA polymerase prevents on-target damage and enhances precision of CRISPR editing. Preprint at https://doi.org/10.1101/2023.01.10.523496 (2023).
Yoo, K. W., Yadav, M. K., Song, Q., Atala, A. & Lu, B. Targeting DNA polymerase to DNA double-strand breaks reduces DNA deletion size and increases templated insertions generated by CRISPR/Cas9. Nucleic Acids Res. 50, 3944–3957 (2022).
Google Scholar
Sharon, E. et al. Functional genetic variants revealed by massively parallel precise genome editing. Cell 175, 544–557.e16 (2018).
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
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
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
Chen, P. J. & Liu, D. R. Prime editing for precise and highly versatile genome manipulation. Nat. Rev. Genet. 24, 161–177 (2023).
Google Scholar
Berdis, A. J. Mechanisms of DNA polymerases. Chem. Rev. 109, 2862–2879 (2009).
Google Scholar
Johansson, E. & Dixon, N. Replicative DNA polymerases. Cold Spring Harb. Perspect. Biol. 5, a012799 (2013).
Google Scholar
Ponnienselvan, K. et al. Addressing the dNTP bottleneck restricting prime editing activity. Preprint at https://doi.org/10.1101/2023.10.21.563443 (2023).
Egli, M. & Manoharan, M. Chemistry, structure and function of approved oligonucleotide therapeutics. Nucleic Acids Res. 51, 2529–2573 (2023).
Google Scholar
Chandler, M. et al. Breaking and joining single-stranded DNA: the HUH endonuclease superfamily. Nat. Rev. Microbiol. 11, 525–538 (2013).
Google Scholar
Lovendahl, K. N., Hayward, A. N. & Gordon, W. R. Sequence-directed covalent protein-DNA linkages in a single step using HUH-tags. J. Am. Chem. Soc. 139, 7030–7035 (2017).
Google Scholar
Tompkins, K. J. et al. Molecular underpinnings of ssDNA specificity by Rep HUH-endonucleases and implications for HUH-tag multiplexing and engineering. Nucleic Acids Res. 49, 1046–1064 (2021).
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, 54 (2018).
Google Scholar
Klenow, H. & Overgaard-Hansen, K. Proteolytic cleavage of DNA polymerase from Escherichia coli B into an exonuclease unit and a polymerase unit. FEBS Lett. 6, 25–27 (1970).
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
Li, L. et al. Multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces. J. Virol. 84, 1674–1682 (2010).
Google Scholar
Chandra, A., Hughes, T. R., Nugent, C. I. & Lundblad, V. Cdc13 both positively and negatively regulates telomere replication. Genes Dev. 15, 404–414 (2001).
Google Scholar
Glustrom, L. W. et al. Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function. Proc. Natl Acad. Sci. USA 115, 10315–10320 (2018).
Google Scholar
Smiley, A. T. et al. Watson–Crick base-pairing requirements for ssDNA recognition and processing in replication-initiating HUH endonucleases. mBio 14, e02587-22 (2023).
Google Scholar
Lawyer, F. C. et al. High-level expression, purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated form deficient in 5′ to 3′ exonuclease activity. Genome Res. 2, 275–287 (1993).
Google Scholar
Blanco, L. et al. Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication. J. Biol. Chem. 264, 8935–8940 (1989).
Google Scholar
Esteban, J. A., Soengas, M. S., Salas, M. & Blanco, L. 3′ → 5′ exonuclease active site of phi 29 DNA polymerase. Evidence favoring a metal ion-assisted reaction mechanism. J. Biol. Chem. 269, 31946–31954 (1994).
Google Scholar
Thyme, S. B., Akhmetova, L., Montague, T. G., Valen, E. & Schier, A. F. Internal guide RNA interactions interfere with Cas9-mediated cleavage. Nat. Commun. 7, 11750 (2016).
Google Scholar
Ponnienselvan, K. et al. Reducing the inherent auto-inhibitory interaction within the pegRNA enhances prime editing efficiency. Nucleic Acids Res. 51, 6966–6980 (2023).
Google Scholar
Zhang, W. et al. Enhancing CRISPR prime editing by reducing misfolded pegRNA interactions. eLife 12, RP90948 (2024).
Google Scholar
Nelson, J. W. et al. Engineered pegRNAs improve prime editing efficiency. Nat. Biotechnol. 40, 402–410 (2022).
Google Scholar
Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635–5652.e29 (2021).
Google Scholar
Ferreira da Silva, J. et al. Prime editing efficiency and fidelity are enhanced in the absence of mismatch repair. Nat. Commun. 13, 760 (2022).
Google Scholar
Lahue, R. S., Au, K. G. & Modrich, P. DNA mismatch correction in a defined system. Science 245, 160–164 (1989).
Google Scholar
Su, S. S., Lahue, R. S., Au, K. G. & Modrich, P. Mispair specificity of methyl-directed DNA mismatch correction in vitro. J. Biol. Chem. 263, 6829–6835 (1988).
Google Scholar
Mathis, N. et al. Machine learning prediction of prime editing efficiency across diverse chromatin contexts. Nat. Biotechnol. https://doi.org/10.1038/s41587-024-02268-2 (2024).
Mathis, N. et al. Predicting prime editing efficiency and product purity by deep learning. Nat. Biotechnol. 41, 1151–1159 (2023).
Google Scholar
Paquet, D. et al. Efficient introduction of specific homozygous and heterozygous mutations using CRISPR/Cas9. Nature 533, 125–129 (2016).
Google Scholar
Doman, J. L. et al. Phage-assisted evolution and protein engineering yield compact, efficient prime editors. Cell 186, 3983–4002.e26 (2023).
Google Scholar
Petri, K. et al. CRISPR prime editing with ribonucleoprotein complexes in zebrafish and primary human cells. Nat. Biotechnol. 40, 189–193 (2022).
Google Scholar
Grünewald, J. et al. Engineered CRISPR prime editors with compact, untethered reverse transcriptases. Nat. Biotechnol. 41, 337–343 (2023).
Google Scholar
Li, X. et al. Highly efficient prime editing by introducing same-sense mutations in pegRNA or stabilizing its structure. Nat. Commun. 13, 1669 (2022).
Google Scholar
Ricchetti, M. & Buc, H. E. coli DNA polymerase I as a reverse transcriptase. EMBO J. 12, 387–396 (1993).
Google Scholar
Krzywkowski, T., Kühnemund, M., Wu, D. & Nilsson, M. Limited reverse transcriptase activity of phi29 DNA polymerase. Nucleic Acids Res. 46, 3625–3632 (2018).
Google Scholar
Kim, D. Y., Moon, S. B., Ko, J.-H., Kim, Y.-S. & Kim, D. Unbiased investigation of specificities of prime editing systems in human cells. Nucleic Acids Res. 48, 10576–10589 (2020).
Google Scholar
Yu, Z. et al. PEAC-seq adopts Prime Editor to detect CRISPR off-target and DNA translocation. Nat. Commun. 13, 7545 (2022).
Google Scholar
Liang, S.-Q. et al. Genome-wide detection of CRISPR editing in vivo using GUIDE-tag. Nat. Commun. 13, 437 (2022).
Google Scholar
Liang, S.-Q. et al. Genome-wide profiling of prime editor off-target sites in vitro and in vivo using PE-tag. Nat. Methods 20, 898–907 (2023).
Google Scholar
Bae, S., Park, J. & Kim, J.-S. Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
Google Scholar
Kamtekar, S. et al. Insights into strand displacement and processivity from the crystal structure of the protein-primed DNA polymerase of bacteriophage phi29. Mol. Cell 16, 609–618 (2004).
Google Scholar
Rodríguez, I. et al. A specific subdomain in phi29 DNA polymerase confers both processivity and strand-displacement capacity. Proc. Natl Acad. Sci. USA 102, 6407–6412 (2005).
Google Scholar
de Vega, M., Lázaro, J. M., Mencía, M., Blanco, L. & Salas, M. Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs. Proc. Natl Acad. Sci. USA 107, 16506–16511 (2010).
Google Scholar
Povilaitis, T., Alzbutas, G., Sukackaite, R., Siurkus, J. & Skirgaila, R. In vitro evolution of phi29 DNA polymerase using isothermal compartmentalized self replication technique. Protein Eng. Des. Sel. 29, 617–628 (2016).
Google Scholar
Ong, J., Tanner, N., Zhang, Y., Bei, Y. & Potapov, V. Variant DNA polymerases having improved properties and method for improved isothermal amplification of a target DNA. US Patent 11,371,028 (2021).
Plaper, T. et al. Coiled-coil heterodimers with increased stability for cellular regulation and sensing SARS-CoV-2 spike protein-mediated cell fusion. Sci. Rep. 11, 9136 (2021).
Google Scholar
Lainšček, D. et al. Coiled-coil heterodimer-based recruitment of an exonuclease to CRISPR/Cas for enhanced gene editing. Nat. Commun. 13, 3604 (2022).
Google Scholar
Liu, B. et al. Targeted genome editing with a DNA-dependent DNA polymerase and exogenous DNA-containing templates. Nat. Biotechnol. https://doi.org/10.1038/s41587-023-01947-w (2023).
Google Scholar
Trojan, J. et al. Functional analysis of hMLH1 variants and HNPCC-related mutations using a human expression system. Gastroenterology 122, 211–219 (2002).
Google Scholar
Roberts, T. C., Langer, R. & Wood, M. J. A. Advances in oligonucleotide drug delivery. Nat. Rev. Drug Discov. 19, 673–694 (2020).
Google Scholar
Pan, W. et al. DNA polymerase preference determines PCR priming efficiency. BMC Biotech. 14, 10 (2014).
Google Scholar
Choi, J. et al. Precise genomic deletions using paired prime editing. Nat. Biotechnol. 40, 218–226 (2022).
Google Scholar
Jiang, T., Zhang, X. O., Weng, Z. & Xue, W. Deletion and replacement of long genomic sequences using prime editing. Nat. Biotechnol. 40, 227–234 (2022).
Google Scholar
Anzalone, A. V. et al. Programmable deletion, replacement, integration and inversion of large DNA sequences with twin prime editing. Nat. Biotechnol. 40, 731–740 (2022).
Google Scholar
Yarnall, M. T. N. et al. Drag-and-drop genome insertion of large sequences without double-strand DNA cleavage using CRISPR-directed integrases. Nat. Biotechnol. 41, 500–512 (2023).
Google Scholar
Zheng, C. et al. Template-jumping prime editing enables large insertion and exon rewriting in vivo. Nat. Commun. 14, 3369 (2023).
Google Scholar
Wang, J. et al. Efficient targeted insertion of large DNA fragments without DNA donors. Nat. Methods 19, 331–340 (2022).
Google Scholar
Gasiunas, G. et al. A catalogue of biochemically diverse CRISPR–Cas9 orthologs. Nat. Commun. 11, 5512 (2020).
Google Scholar
Altae-Tran, H. et al. The widespread IS200/IS605 transposon family encodes diverse programmable RNA-guided endonucleases. Science 374, 57–65 (2021).
Google Scholar
Martín-Alonso, S., Frutos-Beltrán, E. & Menéndez-Arias, L. Reverse transcriptase: from transcriptomics to genome editing. Trends Biotechnol. 39, 194–210 (2021).
Google Scholar
Shuto, Y. et al. Structural basis for pegRNA-guided reverse transcription by a prime editor. Nature 631, 224–231 (2024).
Google Scholar
Yang, L. et al. Efficient delivery of antisense oligonucleotides using bioreducible lipid nanoparticles in vitro and in vivo. Mol. Ther. Nucleic Acids 19, 1357–1367 (2020).
Google Scholar
Farbiak, L. et al. All‐in‐one dendrimer‐based lipid nanoparticles enable precise HDR‐mediated gene editing in vivo. Adv. Mater. 33, 2006619 (2021).
Google Scholar
Dahlman, J. E. et al. Barcoded nanoparticles for high throughput in vivo discovery of targeted therapeutics. Proc. Natl Acad. Sci. USA 114, 2060–2065 (2017).
Google Scholar
Xue, L. et al. High-throughput barcoding of nanoparticles identifies cationic, degradable lipid-like materials for mRNA delivery to the lungs in female preclinical models. Nat. Commun. 15, 1884 (2024).
Google Scholar
Chen, K. et al. Lung and liver editing by lipid nanoparticle delivery of a stable CRISPR–Cas9 RNP. Preprint at https://doi.org/10.1101/2023.11.15.566339 (2023).
Wei, T. et al. Lung SORT LNPs enable precise homology-directed repair mediated CRISPR/Cas genome correction in cystic fibrosis models. Nat. Commun. 14, 7322 (2023).
Google Scholar
Onuma, H., Sato, Y. & Harashima, H. Lipid nanoparticle-based ribonucleoprotein delivery for in vivo genome editing. J. Controlled Release 355, 406–416 (2023).
Google Scholar
Kazlauskas, D., Varsani, A., Koonin, E. V. & Krupovic, M. Multiple origins of prokaryotic and eukaryotic single-stranded DNA viruses from bacterial and archaeal plasmids. Nat. Commun. 10, 3425 (2019).
Google Scholar
Kleinstiver, B. P. et al. Engineered CRISPR–Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Google Scholar
Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).
Google Scholar
Kleinstiver, B. P. et al. Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Google Scholar
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Google Scholar
BBMap. SourceForge https://sourceforge.net/projects/bbmap (2022).
Iseli, C., Ambrosini, G., Bucher, P. & Jongeneel, C. V. Indexing strategies for rapid searches of short words in genome sequences. PLoS One 2, e579 (2007).
Google Scholar
Ferreira da Silva J., et al. Click editing enables programmable genome writing using DNA polymerases and HUH endonucleases. (Dataset. NCBI Sequence Read Archive); https://www.ncbi.nlm.nih.gov/bioproject/PRJNA1015647 (2024).
