Wang, J. Y. & Doudna, J. A. CRISPR technology: a decade of genome editing is only the beginning. Science 379, eadd8643 (2023).
Zhang, J. et al. Non-viral, specifically targeted CAR-T cells achieve high safety and efficacy in B-NHL. Nature 609, 369–374 (2022).
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).
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).
Pandey, S. et al. Efficient site-specific integration of large genes in mammalian cells via continuously evolved recombinases and prime editing. Nat. Biomed. Eng. 9, 22–39 (2025).
Sun, C. et al. Precise integration of large DNA sequences in plant genomes using PrimeRoot editors. Nat. Biotechnol. 42, 316–327 (2024).
Sharma, R. et al. In vivo genome editing of the albumin locus as a platform for protein replacement therapy. Blood 126, 1777–1784 (2015).
Roth, T. L. et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 559, 405–409 (2018).
Carter, A., Brackley, S. M., Gao, J. & Mann, J. P. The global prevalence and genetic spectrum of lysosomal acid lipase deficiency: a rare condition that mimics NAFLD. J. Hepatol. 70, 142–150 (2019).
Kumrah, R. et al. Genetics of severe combined immunodeficiency. Genes Dis. 7, 52–61 (2020).
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Mali, P. et al. RNA-guided human genome engineering via Cas9. Science 339, 823–826 (2013).
Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
Jinek, M. et al. RNA-programmed genome editing in human cells. eLife 2, e00471 (2013).
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).
Tsuchida, C. A. et al. Mitigation of chromosome loss in clinical CRISPR–Cas9-engineered T cells. Cell 186, 4567–4582 (2023).
Porter, D. L., Levine, B. L., Kalos, M., Bagg, A. & June, C. H. Chimeric antigen receptor-modified T cells in chronic lymphoid leukemia. N. Engl. J. Med. 365, 725–733 (2011).
Oh, S. A. et al. High-efficiency nonviral CRISPR/Cas9-mediated gene editing of human T cells using plasmid donor DNA. J. Exp. Med. 219, e20211530 (2022).
Roth, T. L. et al. Non-viral intron knock-ins for targeted gene integration into human T cells and for T-cell selection. Nat. Biomed. Eng. 9, 1309–1319 (2025).
Eyquem, J. et al. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection. Nature 543, 113–117 (2017).
Ozdemirli, M. et al. Indolent CD4+ CAR T-cell lymphoma after cilta-cel CAR T-cell therapy. N. Engl. J. Med. 390, 2074–2082 (2024).
Harrison, S. J. et al. CAR+ T-cell lymphoma post ciltacabtagene autoleucel therapy for relapsed refractory multiple myeloma. Blood 142, 6939–6939 (2023).
Perica, K. et al. CD4+ T-cell lymphoma harboring a chimeric antigen receptor integration in TP53. N. Engl. J. Med. 392, 577–583 (2025).
Rees, H. A., Yeh, W. H. & Liu, D. R. Development of hRad51–Cas9 nickase fusions that mediate HDR without double-stranded breaks. Nat. Commun. 10, 2212 (2019).
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).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Nakajima, K. et al. Precise and efficient nucleotide substitution near genomic nick via noncanonical homology-directed repair. Genome Res. 28, 223–230 (2018).
Chen, X. et al. In trans paired nicking triggers seamless genome editing without double-stranded DNA cutting. Nat. Commun. 8, 657 (2017).
Hyodo, T. et al. Tandem paired nicking promotes precise genome editing with scarce interference by p53. Cell Rep. 30, 1195–1207 (2020).
Ma, M. et al. Efficient generation of mice carrying homozygous double-floxp alleles using the Cas9-Avidin/Biotin-donor DNA system. Cell Res. 27, 578–581 (2017).
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).
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).
Nguyen, D. N. et al. Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency. Nat. Biotechnol. 38, 44–49 (2020).
Gu, B., Posfai, E. & Rossant, J. Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos. Nat. Biotechnol. 36, 632–637 (2018).
Shy, B. R. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat. Biotechnol. 41, 521–531 (2023).
Wimberger, S. et al. Simultaneous inhibition of DNA-PK and Polϴ improves integration efficiency and precision of genome editing. Nat. Commun. 14, 4761 (2023).
Lim, K. H., Huang, H., Pralle, A. & Park, S. Stable, high-affinity streptavidin monomer for protein labeling and monovalent biotin detection. Biotechnol. Bioeng. 110, 57–67 (2013).
Shibata, M. et al. Real-space and real-time dynamics of CRISPR–Cas9 visualized by high-speed atomic force microscopy. Nat. Commun. 8, 1430 (2017).
Yin, J. et al. Optimizing genome editing strategy by primer-extension-mediated sequencing. Cell Discov. 5, 18 (2019).
Wang, C. et al. dCas9-based gene editing for cleavage-free genomic knock-in of long sequences. Nat. Cell Biol. 24, 268–278 (2022).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR–Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Bodai, Z., Bishop, A. L., Gantz, V. M. & Komor, A. C. Targeting double-strand break indel byproducts with secondary guide RNAs improves Cas9 HDR-mediated genome editing efficiencies. Nat. Commun. 13, 2351 (2022).
Charpentier, M. et al. CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair. Nat. Commun. 9, 1133 (2018).
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).
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).
Ren, J. et al. Multiplex genome editing to generate universal CAR T cells resistant to PD1 inhibition. Clin. Cancer Res. 23, 2255–2266 (2017).
Samuelson, C. et al. Multiplex CRISPR/Cas9 genome editing in hematopoietic stem cells for fetal hemoglobin reinduction generates chromosomal translocations. Mol. Ther. Methods Clin. Dev. 23, 507–523 (2021).
Allen, A. G. et al. A highly efficient transgene knock-in technology in clinically relevant cell types. Nat. Biotechnol. 42, 458–469 (2024).
Chavez, M., Rane, D. A., Chen, X. & Qi, L. S. Stable expression of large transgenes via the knock-in of an integrase-deficient lentivirus. Nat. Biomed. Eng. 7, 661–671 (2023).
Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–1019 (2014).
An, J. et al. Enhancement of the viability of T cells electroporated with DNA via osmotic dampening of the DNA-sensing cGAS–STING pathway. Nat. Biomed. Eng. 8, 149–164 (2024).
Xie, K. Efficient non-viral immune cell engineering using circular single-stranded DNA-mediated genomic integration. Nat. Biotechnol. 43, 1821–1832 (2025).
Tou, C. et al. Immune evasive DNA donors and recombinases license kilobase-scale writing. Nature 653, 576–586 (2026).
Roth, T. L. et al. Pooled knockin targeting for genome engineering of cellular immunotherapies. Cell 181, 728–744 (2020).
Witte, I. P. et al. Programmable gene insertion in human cells with a laboratory-evolved CRISPR-associated transposase. Science 388, eadt5199 (2025).
Fell, C. W. et al. Reprogramming site-specific retrotransposon activity to new DNA sites. Nature 642, 1080–1089 (2025).
Pelea, O. et al. Programmable genome editing in human cells using RNA-guided bridge recombinases. Science 391, eadz1884 (2026).
Shi, Y.-j. et al. Quadruple pegRNA enables programmable and efficient large genomic insertion. Nature 654, 272–281 (2026).
Zheng, Y. et al. Efficient in vivo homology-directed repair within cardiomyocytes. Circulation 145, 787–789 (2022).
Liao, H., Wu, J., VanDusen, N. J., Li, Y. & Zheng, Y. CRISPR–Cas9-mediated homology-directed repair for precise gene editing. Mol. Ther. Nucleic Acids 35, 102344 (2024).
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Xu, H. et al. TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells. Nucleic Acids Res. 48, e127 (2020).
Hamilton, J. R. et al. Targeted delivery of CRISPR–Cas9 and transgenes enables complex immune cell engineering. Cell Rep. 35, 109207 (2021).
Blaeschke, F. et al. Modular pooled discovery of synthetic knockin sequences to program durable cell therapies. Cell 186, 4216–4234 (2023).
Hamilton, J. R. et al. In vivo human T cell engineering with enveloped delivery vehicles. Nat. Biotechnol. 42, 1684–1692 (2024).
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, 969–985 (2021).
Riesenberg, S. et al. Simultaneous precise editing of multiple genes in human cells. Nucleic Acids Res. 47, e116 (2019).
Flottmann, F., Pohl, G. M., Gummert, J., Milting, H. & Brodehl, A. A detailed protocol for expression, purification, and activity determination of recombinant SaCas9. STAR Protoc. 3, 101276 (2022).
Hunter, T. L. et al. In vivo CAR T cell generation to treat cancer and autoimmune disease. Science 388, 1311–1317 (2025).
Jacobi, A. M. et al. Simplified CRISPR tools for efficient genome editing and streamlined protocols for their delivery into mammalian cells and mouse zygotes. Methods 121–122, 16–28 (2017).
Ying, Z. et al. A safe and potent anti-CD19 CAR T cell therapy. Nat. Med. 25, 947–953 (2019).
Larson, R. C. et al. CAR T cell killing requires the IFNγR pathway in solid but not liquid tumours. Nature 604, 563–570 (2022).
Shi, J. et al. Lyophilized lymph nodes for improved delivery of chimeric antigen receptor T cells. Nat. Mater. 23, 844–853 (2024).
Künkele, A. et al. Manufacture of chimeric antigen receptor T cells from mobilized cyropreserved peripheral blood stem cell units depends on monocyte depletion. Biol. Blood Marrow Transplant. 25, 223–232 (2019).
Xu, X. et al. IFN-γ-producing Th1-like regulatory T cells may limit acute cellular renal allograft rejection: paradoxical post-transplantation effects of IFN-γ. Immunobiology 222, 280–290 (2017).
Dai, X. et al. Massively parallel knock-in engineering of human T cells. Nat. Biotechnol. 41, 1239–1255 (2023).
Chen, A. X. Y. et al. Rewiring endogenous genes in CAR T cells for tumour-restricted payload delivery. Nature 644, 241–251 (2025).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Liu, Y. et al. PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair. STAR Protoc. 3, 101088 (2022).
Xin, C. et al. Comprehensive assessment of miniature CRISPR–Cas12f nucleases for gene disruption. Nat. Commun. 13, 5623 (2022).
Krzywinski, M. et al. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639–1645 (2009).
Editing Group. Next-Generation Sequencing data of KE. Dataset. Figshare https://doi.org/10.6084/m9.figshare.32358084 (2026).

