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Efficient and precise programmable DNA knock-in without double-strand breaks

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Abstract

Programmable gene knock-in holds substantial promise for treating genetic diseases and advancing cell therapies. However, achieving precise and efficient kilobase-scale DNA fragment integration remains challenging1,2. Here we report CRISPR kilobase-scale nickase-targeting (KNIT) editing for efficient, precise and programmable kilobase-scale DNA insertion without double-strand DNA cleavage, which is enabled through the coupling of a Cas9 nickase with a DNA donor recruiting system. KNIT editing facilitates programmable integration of DNA fragments from 0.7 kb to more than 10 kb and is effective across genomic loci and cell types. It achieves up to 89% efficiency and markedly reduces unintended insertion-deletion mutation (indels) rates, translocations and off-target editing. The system supports repeated insertion editing and multiloci gene knock-in with minimal translocations. Its enhanced version, KNIT editor 2, further improves efficiency via a single transfection. Moreover, in mutant cells with a pathological mutation, KNIT editing restores normal gene expression by inserting a therapeutic gene into a safe harbour locus or its native locus. Notably, KNIT editing enables non-viral and programmable chimeric antigen receptor T cell (CAR-T cell) engineering without double-strand breaks and with clinically relevant efficiencies. Moreover, the engineered CAR-T cells exhibit effective antitumour activity in vitro and in mouse models. Therefore, by achieving programmable and site-specific kilobase-scale DNA insertions without double-strand breaks while reducing unintended outcomes, KNIT editing provides a versatile platform for advancing personalized medicine.

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Fig. 1: Recruiting DNA donors enhances the efficiency of programmable single-nick-based gene knock-in.
Fig. 2: Characterization of KNIT editing regarding large inserts, different loci and evaluation of translocations and off-target editing.
Fig. 3: Optimization of KNIT editing using KE2 and multiloci insertions.
Fig. 4: Integration of therapeutic genes into the AAVS1 and native loci.
Fig. 5: CAR-T cell engineering and gene knock-in in human primary T lymphocytes.

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Data availability

All relevant data supporting the findings of this study are available in the paper and Supplementary Information files of this Article. HTS data are available at Figshare (https://doi.org/10.6084/m9.figshare.32358084)82. Additional datasets are available online (https://www.wanglab-thu.org.cn/knit). Source data are provided with this paper.

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Acknowledgements

We are grateful to members of the Wang Laboratory for discussion and comments during the preparation of the manuscript; and H. Yu and S. Qi for collaboration and discussions on this project.

Funding

This work was funded by the following grants: the Agriculture Science and Technology Major Project (to H.W.), the National Key R&D Program of China (2022YFC3400200 to H.W.), the National Natural Science Foundation of China (32270573 to H.W.), the Tsinghua University Initiative Scientific Research Program, the Independent Research Fund of the State Key Laboratory of Complex, Severe, and Rare Diseases (2025-I-ZD-005), the Tsinghua–Peking Joint Center for Life Sciences, and the Benyuan Charity Fund.

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Author notes

  1. These authors contributed equally: Yanmin Gao, Yu Ma, Kexin Yu, Yintian Liu

Authors and Affiliations

  1. School of Life Sciences, Tsinghua–Peking Joint Center for Life Sciences, Center for Synthetic and Systems Biology, State Key Laboratory of Complex, Severe, and Rare Diseases, Tsinghua University, Beijing, China

    Yanmin Gao  (郜艳敏), Yu Ma  (马宇), Kexin Yu  (于可欣), Yintian Liu  (刘印天), Buming Gu  (顾卜铭), Hao Tang  (唐昊), Wenjie Yan  (燕文杰), Shuangshuang Yang  (杨双双), Jingran Su  (苏敬冉), Xindong Wang  (王新东), Xin Ma  (马昕), Xinming Wang  (王新铭), Fang Wang  (王芳), Qingyang Li  (李青阳), Mengying Liu  (刘梦莹) & Haifeng Wang  (王海峰)

  2. Academy for Advanced Interdisciplinary Studies (AAIS), and Peking University–Tsinghua University–National Institute of Biological Sciences Joint Graduate Program (PTN), Peking University, Beijing, China

    Yintian Liu  (刘印天)

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  1. Yanmin Gao  (郜艳敏)
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  2. Yu Ma  (马宇)
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Contributions

H.W. and Y.G. conceived and designed the study. H.W. supervised the work. Y.G., Y.M. and K.Y. performed molecular and cellular experiments. K.Y. and Y.L. performed the T cell experiments. Y.M., Y.L., H.T. and Q.L. performed HTS analyses. Y.M., Y.L., B.G. and Xinming Wang analysed the data. S.Y., J.S. and Xindong Wang performed the mouse experiments. W.Y. and X.M. performed the genotyping experiments. S.Y., W.Y., F.W., X.M. and M.L. assisted with experiments. H.W., Y.G., Y.M. and Y.L. wrote the manuscript, with input from all authors.

Corresponding author

Correspondence to Haifeng Wang  (王海峰).

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Competing interests

H.W., Y.G. and Y.M. have filed patents for DNA integration related to this manuscript (PCT/CN2025/129171). The other authors declare no competing interests.

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Nature thanks Justin Eyquem, Manuel Gonçalves, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Fig. 1 KNIT editing for DSB-free programmable gene knock-in.

a, Schematic of different strategies tested for CRISPR-mediated DNA insertions at single nicks, including nCas9/sgRNA with unlabeled dsDNA donors (nCas9), nCas9/sgRNA with unlabeled dsDNA donors under NHEJ inhibitor treatment, hRAD51*-nCas9/sgRNA with unlabeled dsDNA donors, or nCas9-mSA/sgRNA with biotinylated donors, respectively. hRAD51* refers to the RAD51 S208E and A209D mutant. Related to Fig. 1a–c. b, Relative insertion efficiency of different systems (nCas9, nCas9 under M3814, AZD-7648, NU7441 and NU7026 treatment, respectively) for inserting EGFP-P2A sequences at the ACTB locus, normalized to control (Ctrl): HEK293T cells treated with nCas9 and sgACTB under DMSO treatment. Related to Fig. 1a strategy (2). c, Schematic of additional strategies tested. Related to Fig. 1d–f. d, Representative confocal images of HEK293T cells 14 days after transfection of the nCas9, KNIT editor 1 (KE1), Cas9 or paired nickases systems for EGFP-P2A insertion at the ACTB locus, respectively. Experiments were replicated independently three times. Scale bars, 50 μm. e,f, Long-term stability of EGFP expression (e) and representative flow cytometry analysis (f) of HEK293T cells after inserting EGFP-P2A into the ACTB locus using Cas9 and KE1 on day 14 and 54 after transfection. g, Schematic of HTS library preparation for quantifying the indel rates in uninserted alleles using nested PCR. HA-L, left homology arm; HA-R, right homology arm. h,i, The percentages of EGFP+ HEK293T cells (h) and indel rates in uninserted alleles (i) after inserting EGFP-P2A at the ACTB locus using Cas9-mSA with unlabeled donor or biotinylated donor, respectively. sgNT: non-targeting sgRNA; sgACTB: sgRNA targeting the ACTB locus. For b, e, h and i, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: one-way ANOVA with Tukey’s multiple-comparisons test (b) or unpaired two-tailed Student’s t-test (e,h and i).

Source data

Extended Data Fig. 2 Investigating the molecular mechanisms of KNIT editing and larger DNA insertions.

a, Schematic of HA-altered donor variants (top) and the percentages of EGFP+ HEK293T cells (bottom) at day 14 post-transfection with nCas9-mSA for EGFP-P2A insertion at the ACTB locus. Donors contain original HA or one of three HA variants: non-homologous arms of equal length (NH-A), complete HA deletion (No HA), or short 25-bp HAs (25 bp HA). b, Schematic (top left) of shRNA-mediated knockdown of key DNA repair proteins (PARP1, BRCA2, and RAD51), their relative expression after knockdown with two independent shRNAs (top right), and fold changes (bottom) in knock-in efficiency following Cas9- or KE1–mediated EGFP-P2A insertion at the ACTB locus under each condition. Relative expression and fold changes were normalized to HEK293T cells treated with non-targeting shRNA (shNT). The P values comparing each experimental condition with the shNT group are shown in the graph. c, Schematic (left) for cell-cycle synchronization using thymidine and nocodazole and fold changes of knock-in efficiency (right) following Cas9- or KE1-mediated insertion of EGFP-P2A at the ACTB locus in HEK293T cells, with or without cell-cycle inhibitors. Fold changes were normalized to the DMSO only group (grey). d,e, Representative flow cytometry analysis of HEK293T cells after inserting a 5.7 kb DNA fragment containing EGFP sequence (d) or inserting a 10.5 kb DNA fragment containing tdTomato sequence (e) into the ACTB locus. Control: HEK293T cells treated with non-targeting sgRNA. Related to Fig. 2a–c. For a-c, Bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: one-way ANOVA with Tukey’s multiple-comparisons test.

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Extended Data Fig. 3 HTS analysis of on-target outcomes and off-target insertions.

a, The workflow of the modified PEM-seq for analyzing on-target outcomes (e.g. desired insertion, indels and translocations), using one-round primer extension from the genomic regions near the sgRNA targeting site. Related to Fig. 2g,h. b,c, Bar graphs comparing the percentages of undesired insertions (b) and deletions (c) in total edited alleles at the ACTB locus in HEK293T cells after inserting EGFP-P2A using Cas9 and KE1, respectively. Bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test. d, The workflow of the modified PEM-seq for analyzing genome-wide off-target insertions. One-round primer extension was performed using capture primers designed to target the insert region (e.g., EGFP gene). After capture and purification, UMI sequences were added via ligation. Then nested PCR was performed using nested primers containing sequencing adaptors, followed by amplification and HTS. Related to Fig. 2i. UMI: unique molecular identifier.

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Extended Data Fig. 4 KNIT editing enables repeated editing to increase insertion efficiency.

a, Schematic of repeated insertions at a specific locus via a second (2nd) transfection of Cas9 or KE1. Cas9 generates indels that hinder the original sgRNA binding during the 2nd transfection. b,c, Representative flow cytometry analysis (b) and percentages of EGFP+ HEK293T cells (c) following Cas9- or KE1-mediated EGFP-P2A insertion into the ACTB locus after the first (1st) or second (2nd) transfection. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. d,e, Representative flow cytometry analysis (d) and the percentages of EGFP+ HEK293T cells (e) following Cas9- or KE1-mediated P2A-EGFP insertion at the H2B locus after the first (1st) or second (2nd) transfection. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. f, Cas9-induced off-target insertion sites (left) and bar graph (right) showing off-target insertion efficiencies following repeated editing with Cas9 or KE1 for EGFP-P2A insertion at the ACTB locus. OT1-OT4: Cas9-mediated off-target insertion sites identified by genome-wide analysis, based on a previously reported 1% threshold. Red letters indicate sgRNA mismatch sites within the off-target DNA sequences. See Extended Data Fig. 3d for method details. For c, e, and f, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test.

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Extended Data Fig. 5 Screening strategies for improving efficiency of KNIT editing.

a, Schematic of kilobase-scale DNA insertions by KNIT editing under NHEJ inhibitor treatment. Related to Fig. 3a. b, Bar graphs comparing the relative efficiencies of KE1-mediated EGFP-P2A (798 bp) insertion at the ACTB locus (left) or SA-T2A-EGFP-bGH (1100 bp) insertion at the AAVS1 locus (right) under treatment of different NHEJ inhibitors (M3814, AZD-7648, NU7441 or NU7026), normalized to cells treated with DMSO (Ctrl). c, Other tested strategies for improving KNIT editing insertion efficiency, including fusing nCas9 (D10A)-mSA with hRAD51* (S208E and A209D), CtIP and 53BP1-DN1S. d, Relative insertion efficiencies of different protein-recruiting systems (hRAD51*-nCas9-mSA, CtIP-nCas9-mSA, DN1S-nCas9-mSA) for inserting EGFP sequences at the ACTB (left), H2B (middle) and AAVS1 (right) loci in HEK293T cells, normalized to the efficiency of KE1. e, Representative flow cytometry analysis of EGFP+ HEK293T cells following Cas9-, KE1-, or KE2-mediated SA-T2A-EGFP-bGH (1100 bp) insertion at the AAVS1 locus. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. For b and d, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: one-way ANOVA with Tukey’s multiple-comparisons test.

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Extended Data Fig. 6 KNIT editor 2 improved insertion efficiency at different genomic loci.

a,b, Schematic (left) and bar graph (right) comparing the percentages of EGFP+ or mCherry+ HEK293T cells with P2A-EGFP insertion at the H2B locus (a), or mCherry-P2A insertion at the RAB11A locus (b), using Cas9, KE1 or KE2. c,d, Representative flow cytometry analysis of HEK293T cells after inserting P2A-EGFP into the H2B locus (c) or mCherry-P2A into the RAB11A locus (d) using Cas9, KE1 or KE2, respectively. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. e,f, Bar graphs comparing the indel rates in uninserted alleles at the H2B (e) locus and RAB11A (f) locus in HEK293T cells. Cells were treated with Cas9, KE1, or KE2 for inserting P2A-EGFP into the H2B locus (e) or mCherry-P2A into the RAB11A locus (f), respectively. For a, b, e, and f, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test (a and b) or one-way ANOVA with Tukey’s multiple-comparisons test (e and f).

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Extended Data Fig. 7 Knock-in efficiencies across different genomic loci and cell types.

a, Bar graphs comparing the percentages of EGFP+ or mCherry+ HEK293T cells generated by gene knock-in at distinct genomic loci using Cas9, Cas9-mSA, nCas9, KE1, or KE2, with EGFP-P2A (798 bp) inserted at the ACTB locus (left), mCherry-P2A (789 bp) at the RAB11A locus (middle) or SA-T2A-EGFP-bGH (1100 bp) at the AAVS1 locus (right). Biotinylated dsDNA donors were used in the Cas9-mSA, KE1, and KE2 groups. b, Bar graphs comparing the percentages of EGFP+ or mCherry+ K562 cells generated by gene knock-in at distinct genomic loci using Cas9, Cas9-mSA, nCas9, KE1, or KE2, with EGFP-P2A inserted at the ACTB locus (left), mCherry-P2A at the RAB11A locus (middle) or SA-T2A-EGFP-bGH at the AAVS1 locus (right). Biotinylated dsDNA donors were used in the Cas9-mSA, KE1, and KE2 groups. c, Bar graphs comparing the percentages of fluorescent HEK293T cells with 5.7 kb insert (GOI 1-EGFP-P2A) or a 10.5 kb insert (GOI 1-EGFP-GOI 2-tdTomato-P2A) at the ACTB locus using Cas9, Cas9-mSA, nCas9, KE1, or KE2. Biotinylated dsDNA donors were used in the Cas9-mSA, KE1, and KE2 groups. GOI 1: dCas12-VPR. GOI 2: dCas13. d, Bar graphs comparing the percentages of EGFP+ NIH3T3 cells with an SA-T2A-EGFP-bGH insertion at the mouse safe harbor Rosa26 locus using Cas9, Cas9-mSA, nCas9, KE1, or KE2. Biotinylated dsDNA donors were used in the Cas9-mSA, KE1, and KE2 groups. For a-d, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates.

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Extended Data Fig. 8 Simultaneous multi-loci gene knock-in.

a,b, Representative flow cytometry analysis of HEK293T cells after inserting EGFP-P2A into the ACTB locus and/or mCherry-P2A into the RAB11A locus using KE1 (a) or Cas9 (b). c,d, Bar graphs comparing the percentages of EGFP+ and/or mCherry+ HEK293T cells following Cas9-mediated EGFP-P2A insertion at the ACTB locus together with either mCherry-P2A at the RAB11A locus (c) or P2A-mCherry at the H2B locus (d). Green: the percentage of EGFP+ cells, pink: the percentage of mCherry+ cells, blue: the percentage of both EGFP+ and mCherry+ cells. e, Bar graph comparing the indel rates in uninserted alleles at the ACTB (left) or RAB11A (right) locus following single or dual insertions of EGFP-P2A at the ACTB locus and/or mCherry-P2A at the RAB11A locus in HEK293T cells using Cas9 (grey) and KE1 (black). f, Bar graph comparing the percentages of off-target EGFP-P2A (ACTB donor) insertion at the RAB11A locus, and off-target mCherry-P2A (RAB11A donor) insertion at the ACTB locus after simultaneous insertion of EGFP-P2A at the ACTB locus and mCherry-P2A at the RAB11A locus using Cas9 or KE1. g, Schematic (top) and gel electrophoresis (bottom) assessing chromosomal translocations between ACTB and RAB11A loci using Cas9 or KE1, replicated independently three times. Ctrl: untreated HEK293T cells. See Supplementary Fig. 4. sgACTB, sgRAB11A, and sgH2B: sgRNAs targeting the ACTB, RAB11A, H2B loci, respectively. For c-f, n = 3 biological replicates; bars show mean ± s.d. For e,f, dots show individual values; P values: unpaired two-tailed Student’s t-test.

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Extended Data Fig. 9 Efficient integration of a therapeutic sequence (IL2RG) into the AAVS1 locus in HEK293T cells.

a,b, Design of three primers (a) and agarose gel analysis of the PCR amplicons (b) for genotyping single-cell clones to identify cells with IL2RG cDNA insertion (1565 bp) at the target AAVS1 locus, shown for one insertion junction. The insertion-free (800 bp) and insertion-specific (648 bp) amplicons can serve as internal controls for each other. Related to Fig. 4c. Each single-cell clone was assigned a unique identifier. The single-cell clones 15, 23, 37, 41, 45, 47, 49, 57, 67, 85, and 95 in the Cas9-treated group (n = 85 cell clones), and clone 41 in the KE1-treated group (n = 95 cell clones), which show no detectable PCR amplicons at both 800 bp (uninserted amplicons) and 648 bp (inserted amplicons) sizes, were excluded from further analysis (grey). See Supplementary Fig. 4. c, Validation of the KE1-mediated insertion of an IL2RG-expressing cassette at the AAVS1 locus by Sanger sequencing of insertion-specific amplicons. d,e, Design of three primers (d) and agarose gel analysis of the PCR amplicons (e) for genotyping single-cell clones to identify cells with IL2RG cDNA insertion at the AAVS1 locus, shown for the other insertion junction. The two insertion-free (1009 bp) and insertion-specific (609 bp) amplicons can serve as internal controls for each other. Related to Fig. 4c. The single-cell clones 15, 23, 37, 41, 45, 47, 49, 57, 67, 85, and 95 in the Cas9-treated group (n = 85 cell clones), and 41 in the KE1-treated group (n = 95 cell clones), which show no detectable PCR amplicons at both 1009 bp (uninserted amplicons) and 609 bp (inserted amplicons) sizes, were excluded from further analysis (grey). See Supplementary Fig. 4. f, Validation of the KE1-mediated insertion of an IL2RG-expressing cassette at the AAVS1 locus by Sanger sequencing of insertion-specific PCR amplicons.

Extended Data Fig. 10 KNIT editing for gene knock-in in T cells and CAR-T engineering.

a, EGFP knock-in efficiency at the ACTB locus in Jurkat E6-1 cells using Cas9 or KE1 RNPs. b, Live (left) and EGFP+ (right) T cell numbers following EGFP-P2A insertion at the ACTB locus in primary T cells 3–5 days after electroporation with Cas9 or KE1 RNPs. c, Long-term stability of EGFP integration at the ACTB locus by KNIT editing, showing EGFP+ percentages (left) and representative flow cytometry plots (right) in sorted populations on days 10, 17, and 24 post-electroporation. d-f, On-target indel rates in uninserted alleles (d), and relative IFNB1 (e) and NOXA (f) expression after EGFP-P2A insertion at the ACTB locus using Cas9 or KE1 RNPs. RT-qPCR was performed 24 h post-electroporation and normalized to the Cas9 group. g-i, Representative flow cytometry (g), live and CAR+ primary T cell numbers (h), and representative confocal images (i) following anti-CD19 CAR insertion at the ACTB locus using Cas9 or KE1 RNPs. Flow cytometry with surface staining was performed with equal fractions of each sample at day 8 after electroporation. Related to Fig. 5f. Scale bars (i), 100 μm. j, CAR-T cell numbers (day 8) following CAR insertion at the ACTB locus using KE1 RNP, KE1 mRNA or KE2 mRNA. Related to Fig. 5h. k, Long-term stability of CAR integration at the ACTB locus by KNIT editing, showing CAR+ percentages (left) and representative flow cytometry plots (right) in sorted CAR-T cells on days 19 and 26 post-electroporation. CAR+ cells were sorted on day 12. l, Off-target insertion rates after CAR integration at the ACTB locus using Cas9 or KE. OT1-OT3: Cas9 off-target insertion sites shown in Fig. 2i. OT1 and OT3 were most prominent in Cas9-mediated CAR-T engineering. m, Circos plots showing chromosomal translocation rates following CAR insertion at the ACTB locus in T cells using Cas9 or KE. ND: Not detected. n, Relative T cell numbers 8 days after electroporation of Cas9 mRNA, KE1 mRNA and KE2 mRNA for CAR insertion at the TRAC locus, normalized to Cas9. o, Time-course of relative EGFP fluorescence intensity of CD19-EGFP-expressing HEK293T target cells co-cultured with indicated CAR-T cells and untreated T cells. Target cell only: CD19-EGFP HEK293T cells without T cells. p, Quantification of fluorescence intensity variance of CAR expression in LV-EF1α-CD19BBz CAR T cells, ACTB-CD19BBz CAR-T cells and TRAC-CD19BBz CAR-T cells. CV: coefficient of variation. For c, g and k: control: untreated T cells. For a-f, h and j-p, bars/values show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test (a, b, d, e, f, and h) or one-way ANOVA with Tukey’s multiple-comparisons test (j, n and p). All experiments were replicated independently at least three times.

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Gao, Y., Ma, Y., Yu, K. et al. Efficient and precise programmable DNA knock-in without double-strand breaks. Nature (2026). https://doi.org/10.1038/s41586-026-10819-7

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