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Non-viral targeted integration of large DNA in primary human T cells independent of double-stranded DNA breaks

Nature Biomedical Engineering (2026) Cite this article

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Abstract

Non-viral targeted integration of large DNA cargoes into human primary T cells typically requires the induction of genomic double-strand breaks (DSBs), a process associated with cytotoxicity and potential tumorigenic chromosomal abnormalities. Here we report PRIME-In, a novel genome-editing platform that uses a prime editing-engineered donor template coupled with either single (PRIME-In 1.0) or paired (PRIME-In 2.0) genomic nicks to enable precise integration of substantial DNA payloads into human cells without reliance on DSB repair pathways. Compared with traditional DSB-dependent methods, PRIME-In demonstrates markedly enhanced editing efficiency and specificity while eliminating detectable on-target and off-target chromosomal aberrations. Subsequent refinement of reagent composition and delivery protocols enabled PRIME-In-mediated engineering of primary human T cells with minimal toxicity, achieving up to 50% integration efficiency for a 3-kb CAR construct. These advances establish PRIME-In as a transformative platform for streamlining the non-viral production of genome-edited T cells, offering substantial potential for T cell-based immunotherapies.

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Fig. 1: PRIME-In allows for DSB-independent, high-efficiency targeted transgene integration in human cells.
The alternative text for this image may have been generated using AI.
Fig. 2: Optimization of the PRIME-In design.
The alternative text for this image may have been generated using AI.
Fig. 3: Comprehensive analysis of the KI outcomes by HTS.
The alternative text for this image may have been generated using AI.
Fig. 4: PRIME-In 2.0 mediates high-efficiency targeted KI in primary T cells.
The alternative text for this image may have been generated using AI.
Fig. 5: HCMV UL36/37x1 promotes PRIME-In 2.0-mediated T cell engineering by preventing cell death upon DNA challenge.
The alternative text for this image may have been generated using AI.
Fig. 6: PRIME-In 2.0-engineered CAR T cells are phenotypically functional both in vitro and in vivo.
The alternative text for this image may have been generated using AI.

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Data availability

The NGS sequencing data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under BioProject accession number PRJNA1241761. Raw data generated by ddPCR and flow cytometry are available via Figshare at https://doi.org/10.6084/m9.figshare.31440034 (ref. 72). Source data are provided with this paper.

Code availability

All custom code used for NGS analysis in this study is publicly available via GitHub at https://github.com/GUOSHU-COOL/PRIME-In_for_T_cell_engineering.

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Acknowledgements

We thank X. Yang and Q. Meng for technical assistance with flow cytometry. We thank Z. Liu, J. Yang and M. Yuan for assistance with plasmid construction and cell culture. We thank C. Xie for mouse nursing. This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (XDC0200000 to W.L. and H.W., and XDA0510400 to C.W. and J.W.); the National Natural Science Foundation of China (32225030 and 82488301 to W.L., 32401248 to C.W. and 32425035 to H.W.); the National Key Research and Development Program (2024YFA0917300 to W.L. and N.T., and 2024YFA1107200 to C.W.); the CAS Project for Young Scientists in Basic Research (YSBR-012 to W.L.); the Beijing Natural Science Foundation (Z230011 to W.L.); the Agriculture Science and Technology Major Project (to W.L. and H.W.); the Project Incubation Fund of the Beijing Institute for Stem Cell and Regenerative Medicine (2025FH108 to C.W.); the Initiative Scientific Research Program, Institute of Zoology, Chinese Academy of Sciences (2023IOZ0204 to H.W. and 2023IOZ0101 to W.L.). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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Author notes

  1. These authors contributed equally: Sen Fang, Na Tang, Yiyun Li, Shuyu Guo, Yangcan Chen.

  2. These authors jointly supervised this work: Haoyi Wang, Chenxin Wang, Wei Li.

Authors and Affiliations

  1. State Key Laboratory of Organ Regeneration and Reconstruction, Institute of Zoology, Chinese Academy of Sciences, Beijing, China

    Sen Fang, Na Tang, Yiyun Li, Shuyu Guo, Yangcan Chen, Xin Wang, Xueke Li, Yanping Hu, Jiawei Hao, Jun Wu, Qi Zhou, Haoyi Wang, Chenxin Wang & Wei Li

  2. University of Chinese Academy of Sciences, Beijing, China

    Sen Fang, Yiyun Li, Shuyu Guo, Xin Wang, Xueke Li, Jiawei Hao, Qi Zhou, Haoyi Wang & Wei Li

  3. Beijing Institute for Stem Cell and Regenerative Medicine, Beijing, China

    Na Tang, Yangcan Chen, Jun Wu, Qi Zhou, Haoyi Wang, Chenxin Wang & Wei Li

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Contributions

C.W., W.L. and H.W. designed the project. S.F., C.W. and Y.L. performed the experiments in HEK293T, HeLa and K562 cell lines. N.T., C.W. and J.H. performed the experiments in primary human T cells. C.W. and Y.L. performed the experiments in embryonic stem cells. C.W., S.F., Y.L., Y.C. and X.L. constructed the plasmids and mRNAs and performed genotyping. C.W. and Y.L. constructed the NGS library. C.W., S.G., Y.C., X.W. and Y.H. developed the sequencing analysis methods. W.L., C.W., Q.Z., H.W. and J.W. provided resources and overseeing. C.W. wrote the paper with input from all authors.

Corresponding authors

Correspondence to Haoyi Wang, Chenxin Wang or Wei Li.

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Extended data

Extended Data Fig. 1 Length of the RT template influences the efficiency of PRIME-In-mediated targeted KI.

a, Scatter plots of FACS analysis showing frequencies of EGFP + HEK293T cells after PRIME-In 1.0-mediated targeted KI with pegRNAs containing various RT template lengths across four genomic loci. b–e, Editing efficiency statistics of PRIME-In 1.0-mediated targeted KI with pegRNAs containing various RT template lengths across four genomic loci: HEK4 (b), HEK3 (c), LSP1 (d) and VEGFA (e), as measured by the frequency of EGFP + HEK293T cells. Three independent biological replicates were performed. The results are presented as the mean ± SD. Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance. f, Diagram of a PCR-based method for validation of primed microhomology (PMH) on primed donors. The prime editor (PE) generates a single-stranded PMH at the nick site on the donor, which can be amplified by PCR with specific primers. g, Detection of the PMHs generated by PE/HEK3-pegRNA and PE/VEGFA-pegRNA using PCR. Primers RT-P1/HEK3-RT-P2 and RT-P1/VEGFA-RT-P2 amplify PCR products of specific sizes (268 bp) on primed donors. This experiment was repeated independently three times with similar results. Representative gel images from one experiment are shown. h, Sanger sequencing of the PCR products confirmed pegRNA-templated elongation of PMHs at nicked sites on primed donors. PBS, primer binding site; RT, reverse transcriptase; PMH, primed microhomology.

Source data

Extended Data Fig. 2 Different forms of PRIME-In 2.0 designs.

a–c, Schematics of different nicking/priming strategies for targeted transgene integration with PRIME-In 2.0. In PRIME-In 2.0 method, an extra nicking on the complementary strand is introduced to enhance transgene integration (a). Alternative designs include: genomic double nicking with double donor priming (b) and genomic priming with single donor priming (c). d, Editing efficiencies for targeted KI of an EF1α-EGFP reporter with three PRIME-In designs across four genomic loci, as measured by frequencies of EGFP + HEK293T cells. Data represent three independent biological replicates. The results are presented as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance.

Source data

Extended Data Fig. 3 Effects of spacer length, RHA length and transgene size on PRIME-In 2.0-mediated targeted integration.

a, Diagram of PRIME-In 2.0-mediated targeted integration with various spacer lengths between the two genomic nicking sites. b and c, Editing efficiencies for targeted integration of an EF1α-EGFP reporter with various spacer lengths at the AAVS1 (b) and HEK4 (c) loci. d, Diagram of PRIME-In 2.0-mediated targeted integration of two different transgenes with primed donors of various RHA lengths at AAVS1 locus. e and f, Editing efficiencies for targeted integration of an EF1α-EGFP reporter (e) and a CAG-PEmax-mCherry reporter (f) with primed donors of various RHA lengths at AAVS1 locus. The editing efficiencies were measured by frequencies of EGFP+ or mCherry+ HEK293T cells 14 days after transfection. Data are from three independent biological replicates. The results are presented as the mean ± SD. Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance.

Source data

Extended Data Fig. 4 PRIME-In mediates efficient targeted integration at therapeutically relevant loci.

a, Editing efficiencies for targeted integration of an EF1α-EGFP reporter with PRIME-In 2.0 across a variety of therapeutically relevant loci, as measured by frequencies of EGFP + HEK293T cell 14 days after transfection. Four to five pairs of sites at each gene locus were evaluated. Three independent biological replicates were performed and the results are shown as the mean ± SD. No statistical comparisons were performed for this screening data. b, Pearson correlation analysis of PRIME-In-mediated integration efficiencies across 48 genomic loci versus PMH GC contents. Integration efficiencies were positively correlated with PMH GC content. c–e, Pearson Correlation analysis of PRIME-In-mediated integration efficiencies across 48 genomic loci and histone marker signals extracted and processed from ChIP-seq data deposited in ENCODE. Integration efficiencies were positively correlated with active histone markers, H3K27ac and H3K4me3, and negatively correlated with heterochromatin marker H3K9me3. Pearson correlation coefficients (r) and P value for each analysis are labelled on the scatter plot. In all cases, P < 0.05 except between PRIME-In and H3K4me3 signal. Integration efficiencies from three individual replicates, measured by frequencies of EGFP+ cells, were averaged and used for analysis. The ChIP-seq signal of each histone modification within a ± 1 kb window centred around the target site was extracted and processed from HEK293T datasets. Statistical significance was calculated using Student’s unpaired two-tailed t-test.

Source data

Extended Data Fig. 5 Comparison of the editing efficiencies of various integration methods at AAVS1 locus.

a, Schematics of various KI methods for targeting an EF1α-EGFP transgene into AAVS1 locus. Traditional HDR donor is a covalent circular plasmid, which harbors long-range (~800 bp) homologous arms flanking the transgene. HMEJ donor is a intracellularly linearized double-stranded DNA template, which harbors short-range (~50 bp) homologous arms flanking the transgene. The donor for the dsCTS-based method is a double-stranded DNA template containing Cas9 target sequences (CTSs) flanking homologous arms on each side. The donor for the ssCTS-based method is a single-stranded DNA template with hybrid CTSs flanked by homologous arms. All aforementioned methods mediate targeted integrations at genomic DSB sites, whereas eePASSIGE, PASTE and PRIME-In mediate targeted integrations at genomic nick sites without introducing DSBs. b, Gel analysis of prepared dsCTS and ssCTS donors. Molecular weight markers (bp) are indicated on the left of the gel image. c, Editing efficiencies of various KI methods for targeting an EF1α-EGFP transgene at AAVS1 locus, as measured by frequencies of EGFP + HEK293T cells 14 days after transfection. Data are from three independent biological replicates. The results are presented as the mean ± SD. Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test.

Source data

Extended Data Fig. 6 Genome-wide profiling of on-target and off-target editing outcomes.

a, Deletion length distribution of AAVS1 bait HTS events following HMEJ-, PRIME-In 1.0-, and PRIME-In 2.0-mediated targeted KI are plotted on a log scale. b, Representative deletion events following PRIME-In 2.0-mediated targeted KI at the AAVS1 locus. The yellow bars indicate the two sgRNA target sites and the dashed line marks the deleted region. The underline shows microhomology. Ref., sequence of the reference assembly. c, Diagram of a DSB-independent “Nick-out” model to explain the deletion events at the target site following PRIME-In 2.0-mediated targeted KI. Double genomic nicking generates two dissociative 3′ DNA ends. The spacer region between the two nick sites is deleted via microhomology-based annealing and repair. d, Integration events triggered by off-target cleavage of nuclease following HMEJ-, eePASSIGE-, PRIME-In 1.0-, and PRIME-In 2.0-mediated targeted KI at AAVS1 locus. e, Summary of identified random integration events with various KI methods targeting AAVS1. f, Representative eeBxb1-induced off-target KI events following eePASSIGE-mediated targeted KI at the AAVS1 locus. The eeBxb1 integrase mediates donor integration at pseudo-attB sites distributed throughout the genome.

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Extended Data Fig. 7 Translocation junction profiles at on-target and predicted off-target sites following targeted KI at AAVS1 locus.

a and b, Circos plots show the genome-wide translocations (orange lines) at two predicted off-target loci: chr6 OT1 (a) and chr19 OT2 (b), following HMEJ-, eePASSIGE-, PRIME-In 1.0-, and PRIME-In 2.0-mediated targeted KI. Colored lines connect the bait site to the prey site. Line colors (orange, from dark to light) indicates high to low hotspot enrichment, respectively.

Extended Data Fig. 8 Screening optimized forms of PE editors for T cell engineering.

a, The constructs of various promoters driving the expression of intein-split PEmax in primary T cells. b and c, Delivery efficiencies (b) and expression intensities (c) after electroporation with 2 μg of plasmids expressing PEmax-Cpart-T2A-mCherry driven by various promoters in primary T cells (from n = 3 independent biological donors). For b and c, The results are presented as the mean ± SEM. Statistical analyses were done using one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance. d, Schematics of split-intein PEmax used in PRIME-In 2.0-mediated targeted KI. e, Editing efficiencies of PRIME-In 2.0-mediated targeted KI with various intein-split PEmax constructs at AAVS1 locus in HEK293T cells. KI efficiencies were measured as the percentage of EGFP+ cells among successfully transfected cells. Three independent biological replicates were performed. The results are presented as the mean ± SD. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance.

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Extended Data Fig. 9 Genomic nicking alleviates cell apoptosis and growth retardation.

a, Diagram of targeted integration of a EGFP reporter into AAVS1 intron 1 in primary T cells with a donor plasmid containing a EF1α-EGFP transgene and a triple-tandem guide RNA expression cassette, in combination with PEmax or its mutant forms: dPEmax and wtPEmax. The dPEmax harbors a D10A mutation and loses DNA cleavage activity. The wtPEmax contains an A840H mutation that restores double-strand cleavage activity of Cas9. We refer to the method using PRIME-In donor plus wtPEmax as DSB_PRIME-In, as it generates genomic DSBs. b–d, Annexin V-PI staining show apoptosis rates of engineered T cells (from n = 3 independent biological donors) 1 day post-electroporation with PRIME-In donor, in combination with various forms of PEmax mRNAs. Electroporation without any editing components or with PRIME-In donor only were used as controls. b, Representivie scatter plots of FACS analysis after Annexin V-PI staining from a single donor. c and d, Statistics of the Annexin V-PI staining results show elevated cell apoptosis when T cells were transfected with PRIME-In donor and wtPEmax, compared with those transfected with PRIME-In donor and PEmax. The results are presented as the mean ± SEM. Statistical significance was calculated using one-way ANOVA with Dunnett’s multiple comparisons test. ns, no significance. e, Representative histogram of Cell Trace Violet staining in T cells 5 days post-electroporation with various editing components. The experiment was independently replicated using primary T cells from n = 3 individual biological donors with similar results.

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Extended Data Fig. 10 The HCMV UL36/UL37×1/A151 cocktial promotes T cell expansion after non-viral genome engineering.

a and b, Delivery efficiency (a) and expression intensity (b) 48 h after electroporation with 1 μg of a plasmid expressing PEmax-Npart, 1 μg of a plasmid expressing PEmax-Cpart-T2A-mCherry, along with various combinations of A151 and mRNAs encoding HCMV components UL31, UL36, and UL37x1 in primary T cells. Plasmid-only treatment was performed as a control. c and d, Percentage of T cells expressing EGFP (c) and fold change in EGFP + T cells over input (d) 7 days post-electroporation with the PRIME-In editing components for targeted integration of the EF1α-EGFP transgene, along with varying doses of UL mRNAs and A151. e and f, T cell viability (e) and fold change in total recovered T cells (f) 7 days after non-viral PRIME-In editing for targeted integration of a 5.8-kb transgene expressing CD19 CAR and EGFP. PRIME-In 2.0-engineered T cells treated with DMSO, apoptosis inhibitors Z-DEVD-FMK, Z-VAD-FMK and innate immune inhibitors BX795, Ruxolitinib during the first two days post-electroporation were examined and compared with the UL36/UL37x1-treated and UL36/UL37x1/A151-treated groups. g and h, Subproportions (g) and differentiation states (h) of CAR T cells after PRIME-In-mediated engineering with or without UL36/UL37x1/A151 cocktail treatment. All experiments were independently replicated using primary T cells from n = 3 individual biological donors. Statistical analyses were done using one-way ANOVA with Dunnett’s multiple comparisons test in a-d and g-h, and two-way ANOVA with Bonferroni’s multiple comparisons test in e and f. The results are presented as the mean ± SEM. ns, no significance. Tn, naive T cells; Tcm, central memory T cells; Tem, effector memory T cells; Teff, effector T cells.

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Statistical source data for Figs. 1–6 and Extended Data Figs. 1–6 and 8–10.

Source Data Extended Data Fig. 1 (download TIF )

Unprocessed gels related to Extended Data Fig. 1.

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Fang, S., Tang, N., Li, Y. et al. Non-viral targeted integration of large DNA in primary human T cells independent of double-stranded DNA breaks. Nat. Biomed. Eng (2026). https://doi.org/10.1038/s41551-026-01671-1

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