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High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails

Abstract

Enhancing CRISPR-mediated site-specific transgene insertion efficiency by homology-directed repair (HDR) using high concentrations of double-stranded DNA (dsDNA) with Cas9 target sequences (CTSs) can be toxic to primary cells. Here, we develop single-stranded DNA (ssDNA) HDR templates (HDRTs) incorporating CTSs with reduced toxicity that boost knock-in efficiency and yield by an average of around two- to threefold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional roughly two- to threefold on average. Our method works across a variety of target loci, knock-in constructs and primary human cell types, reaching HDR efficiencies of >80–90%. We demonstrate application of this approach for both pathogenic gene variant modeling and gene-replacement strategies for IL2RA and CTLA4 mutations associated with Mendelian disorders. Finally, we develop a good manufacturing practice (GMP)-compatible process for nonviral chimeric antigen receptor-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5 × 109 modified cells) exceeding those of conventional approaches.

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Fig. 1: Development of ssCTS templates for high yield knock-in.
Fig. 2: Evaluation of ssCTS design requirements.
Fig. 3: Application of ssCTS knock-in templates across diverse target loci, knock-in constructs and primary human hematopoietic cell types.
Fig. 4: Whole ORF replacement at target genes for therapeutic and diagnostic human T cell editing.
Fig. 5: GMP-compatible process for nonviral CAR-T cell manufacturing.

Data availability

RNA-seq data been deposited in the Gene Expression Omnibus (GEO) under the accession code GSE202909. ATAC-seq data been deposited in the GEO under the accession code GSE130089. Amplicon sequencing data been deposited in the GEO under the accession codes GSE202596 and GSE203349. Other relevant data are available from the corresponding authors upon reasonable request.

Code availability

All code used in this paper will be made available upon reasonable request.

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Acknowledgements

We thank all members of the Marson laboratory for their thoughtful input and technical assistance. We thank S. Dodgson, S. Pyle, F. Urnov, B. Schaar, J. Woo, J. Okano and J. Sawin for their helpful suggestions and generous assistance. This research was supported by the NIAID (grant nos. P01AI138962 and P01AI155393), the UCSF Grand Multiple Myeloma Translational Initiative (MMTI), the Weill Neurohub, the Larry L. Hillblom Foundation (grant no. 2020-D-002-NET) and the Innovative Genomics Institute (IGI). The Marson laboratory has received funds from IGI, the Simons Foundation and the Parker Institute for Cancer Immunotherapy (PICI). A.M. holds a Career Award for Medical Scientists from the Burroughs Wellcome Fund, is an investigator at the Chan Zuckerberg Biohub and is a recipient of The Cancer Research Institute (CRI) Lloyd J. Old STAR grant. B.R.S. was supported by the UCSF Herbert Perkins Cellular Therapy and Transfusion Medicine Fellowship, the CIRM Alpha Stem Cell Clinic Fellowship and the NCATS (grant no. L30TR002983). D.N.N. is supported by National Institutes of Health (NIH) grant nos. L40AI140341 and K08AI153767 and the CIRM Alpha Stem Cell Clinic Fellowship. J.W. and V.S.V. were supported by the UCSF Grand MMTI. F.B. was supported by the Care-for-Rare Foundation and the German Research Foundation (DFG). T.L.R. was supported by the UCSF Medical Scientist Training Program (grant no. T32GM007618), the UCSF Endocrinology Training grant (no. T32 DK007418) and the NIDDK (grant no. F30DK120213). The Eyquem Laboratory has received funding from PICI, Mnemo Therapeutics, Takeda and Cytovia Therapeutics. M.R.M. was supported by the CRI Irvington Fellowship and the Human Vaccines Project Michelson Prize. The UCSF Flow Cytometry Core was supported by grant no. NIH S10 RR028962 and the James B. Pendleton Charitable Trust. This research was made possible by a grant from the California Institute for Regenerative Medicine (grant no. INFR-10361). W.G.P. and C.E.C. acknowledge support from National Science Federation grant no. 1933344 and acknowledge NIH grant no. 1S10OD025096-01A1, which supported the ATM.

Author information

Authors and Affiliations

Authors

Contributions

B.R.S., V.S.V., J.H.E. and A.M. designed the study. B.R.S., V.S.V. and A.H. performed ssCTS experiments. B.R.S. and V.S.V. performed inhibitor experiments. B.R.S. and A.H. performed ORF replacement experiments. B.R.S. and V.S.V. performed GMP-compatible manufacturing experiments. B.R.S., V.S.V., J.-Y.J.C., A.T., J.E., J.H.E., T.G.M. and J.W. designed and performed BCMA-CAR experiments. D.N.N performed HSC experiments. Y.Y.C. and F.B. performed pooled knock-in experiments. S.V. and M.R.M. performed γδ T cell experiments. L.Y. designed and coordinated the large-scale production and downstream purification process of single-stranded DNA repairing template. H.L. supervised the regulatory requirements and quality control methods for ssDNA. W.G.P. and C.E.C. performed AFM studies. T.L.R., E.S., R.Y. and D.W. performed and analyzed amplicon-seq, RNA-seq and ATAC-seq studies. B.R.S., V.S.V. and A.M. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Brian R. Shy or Alexander Marson.

Ethics declarations

Competing interests

A.M. is a compensated cofounder, member of the boards of directors, and a member of the scientific advisory boards of Spotlight Therapeutics and Arsenal Biosciences. A.M. is a cofounder, member of the boards of directors, and a member of the scientific advisory board of Survey Genomics. A.M. is a compensated member of the scientific advisory board of NewLimit. A.M. owns stock in Arsenal Biosciences, Spotlight Therapeutics, NewLimit, Survey Genomics, PACT Pharma and Merck. A.M. has received fees from 23andMe, PACT Pharma, Juno Therapeutics, Trizell, Vertex, Merck, Amgen, Genentech, AlphaSights, Rupert Case Management, Bernstein and ALDA. A.M. is an investor in and informal advisor to Offline Ventures and a client of EPIQ. The Marson laboratory has received research support from Juno Therapeutics, Epinomics, Sanofi, GlaxoSmithKline, Gilead and Anthem. J.E. is a compensated cofounder at Mnemo Therapeutics. J.E. is a compensated scientific advisor to Cytovia Therapeutics. J.E. own stocks in Mnemo Therapeutica and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital. The Eyquem laboratory has received research support from Cytovia Therapeutic and Takeda. J.E. is a holder of patents pertaining to but not resulting from this work. H.L and L.Y. are employees of Genscript Biotech Corporation. J.W. has received consulting fees from Teneobio and Adaptive Biotech. D.N.N. receives consulting fees and sits on the scientific advisory board of Navan Technologies. T.L.R. is a cofounder, holds equity in, and is a member of the Scientific Advisory Board of Arsenal Bioscience. Discounted reagents were provided by Genscript. B.R.S., V.S.V. and A.M. are inventors on patent applications based on the findings described in this paper, a subset of which have been licensed by the University of California. A.H., A.T., W.G.P., Y.Y.C., F.B., E.S., S.V., M.R.M., J.J.C., R.Y., D.W., T.G.M., C.E.C. and J.H.E. declare no competing interests.

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Extended data

Extended Data Fig. 1 Illustration of ssCTS designs.

Detailed illustrations of CTS designs evaluated in the manuscript highlighting the location and orientation of gRNA target sequences (red), 4 bp mismatch (pink), PAM interaction site (yellow), and transgene (blue). (a-j) Illustration of short CD5-HA HDRT designs evaluated in Fig. 1a–c and Extended Data Fig. 2c. (k) Representative illustration of optimal ssCTS design used for large HDRTs throughout the manuscript. CTS = Cas9 Target Site, ssCTS = ssDNA HDRT + CTS sites, HDRT = homology-directed-repair template.

Extended Data Fig. 2 Comparison of CTS template designs.

(a) Diagram of CD5-HA knock-in strategy and control ssDNA HDRTs. (b) Representative flow cytometry plots demonstrating CD5-HA knock-in. (c) Live cell counts for each ssCTS design using a CD5-HA knock-in construct at 160 nM – 4uM concentration. (d-e) Comparison of knock-in efficiency, live cell counts, and knockin cell counts with insertions of increasing size using (d) dsCTS or (e) ssCTS HDRTs targeting the IL2RA gene. Evaluated transgenes encode either GFP (~1.4 kb total HDRT length), an IL2RA-GFP fusion (~2.3 kb total HDRT length), or an IL2RA-GFP fusion plus separate EF1a-mCherry expression cassette (~3.5 kb total HDRT length). Each experiment was performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean. RNP = Ribonucleoprotein, CTS = Cas9 Target Site, ssCTS = ssDNA, HDRT + CTS sites, HDRT = homology-directed-repair template.

Extended Data Fig. 3 Evaluation of ssCTS design and mechanism with large HDRTs.

(a-f) Comparison of different CTS designs with a large ~2.7 kb CD5-HA knock-in construct. (a) Diagram of long CD5-HA knock-in strategy, representative flow cytometry plot, percent knock-in, live cell counts, and knock-in cell yield counts. (b) Comparison of CTS with a gRNA target sequence that is specific for the cognate RNP ( + CD5 CTS), an alternative gRNA sequence (+ IL2RA CTS), a CTS incorporating a PAM site and scrambled gRNA sequence (+ scramble CTS), or an equivalent amount of dsDNA within the 5’ end of the homology arm (+ end protection). (c) Comparison of complementary oligos covering different regions of the CTS and surrounding sequences. Constructs with CTS sites on both 5’ and 3’ end (green bars), 5’ end only (blue bars), or 3’ end only (red bars) are shown on the right panel. (d) Evaluation of varied 5’ ends including different length of buffer sequence upstream of the CTS site. *indicates no data available for the marked column. (e) Comparison of CTS with different numbers of scrambled bases at the 5’ end of the gRNA target sequence using WT or SpyFi Cas9. (f) Length of homology arm that is covered by the complementary oligonucleotide. (g) Evaluation with and without (‘-‘) CTS sites on the 5’ and 3’ end of long ssDNA IL2RA-GFP HDRTs with PAM facing inwards toward the homology arm (‘In’) or outwards away from the homology arm (‘Out’). (h-i) Comparison of knockout and knockin with large CD5-HA (h) or IL2RA-GFP (i) ssDNA and ssCTS HDRTs using RNPs formulated with Cas9 + /- NLS sequences. Each experiment was performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean. RNP = Ribonucleoprotein, CTS = Cas9 Target Site, ssCTS = ssDNA HDRT + CTS sites, PAM = Protospacer Adjacent Motif, HDRT = homology-directed-repair template.

Extended Data Fig. 4 Additional parameters affecting ssCTS knockin and biophysical analysis of RNP interactions with HDRTs.

(a-b) Comparison of knockin efficiency (top) and live cell counts (bottom) +/- PGA using (a) large IL2RA-GFP ssCTS templates or (b) large CD5-HA ssCTS templates (~1.3 kb homology arms). (c) Comparison of knockin efficiency (top) and live cell counts (bottom) with PGA, ssDNA enhancer, or no anionic polymer using a BCMA-CAR ssCTS templates. (d-f) evaluation of (d) indel formation by amplicon sequencing, (e) knockin efficiency with short ssDNA CD5-HA HDRTs, or (f) knockin efficiency with short ssCTS CD5-HA HDRTs (40 nucleotide homology arms) using varied molar amounts of RNP and HDRT. (g) Representative AFM images of gel purified dsCTS or ssCTS templates + /- Cas9 RNPs. Brightness shows the relative height as indicated in by scale bars to right of figure. Background circular forms in all panels are likely residual agarose. Experiments in panels a-f were performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean (a, b, c, e, f) or mean alone (d). RNP = Ribonucleoprotein, CTS = Cas9 Target Site, ssCTS = ssDNA HDRT + CTS sites, HDRT = homology-directed-repair template, AFM = atomic force microscopy.

Extended Data Fig. 5 Arrayed knockin analysis and target locus characteristics.

(a) Comparison of HDRT variations for knock-in constructs targeting a tNGFR marker across 22 different target loci. Shown for each construct are live cell counts, knock-in cell count yields, relative %knock-in and relative knock-in counts compared to dsDNA templates. Data show mean and individual values from 2 independent healthy human blood donors (b-d) Evaluation target locus characteristics in comparison to ssCTS knockin efficiency by (b) Amplicon-Seq, (c) RNA-Seq, and (d) ATAC-Seq methodologies. Knock-in efficiency for panels b-d shows mean from 2 independent healthy human blood donors. Amplicon-Seq, RNA-Seq, and ATAC-seq data in panels b-d were generated 6 independent healthy human blood donors presented as mean + /- SD. Line-of-best-fit and R squared from a simple linear regression are shown for normally distributed data. RNA-seq data was log-transformed prior to linear regression. Spearman r is shown for non-linear correlations as determined by Shapiro-Wilk test (‘distance from cut site’ and ATAC-seq evaluations). Y axis for panels c-d is log 10. In c and d, separate analyses were performed for CD4 + (top) and CD8 + T cells (bottom) for RNA-Seq and ATAC-Seq comparisons. tNGFR = truncated nerve growth factor, MMEJ = microhomology mediated end joining, NHEJ = non-homologous end joining, ATAC = Assay for Transposase-Accessible Chromatin.

Extended Data Fig. 6 Application of ssCTS to diverse primary human hematopoietic cell types.

(a-c) Evaluation of CLTA-mCherry knock-in efficiency and live cell counts 0-10 days postelectroporation in primary human T cells. (a) Live cell counts represented as a percentage of the no electroporation control on day 4 post-electroporation. (b) Knock-in efficiency on day 2-10 post-electroporation. (c) Growth curves for control cells (no electroporation, electroporation only, and Cas9 RNP only) and cells edited with dsCTS or ssCTS HDRTs on day 0-10 post-electroporation. (d) Comparison of knock-in efficiency (top) and live cell counts (bottom) using ssCTS and dsCTS HDRTs (blue line) across a variety of primary human hematopoietic cell types using knockin constructs encoding a CLTA locus mCherry fusion protein. Each experiment was performed with cells from 2 independent healthy human blood donors. Each experiment was performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean (c, d, f) or mean alone (e). CTS = Cas9 target site, dsCTS = dsDNA HDRT + CTS sites, ssCTS = ssDNA HDRT + CTS sites, HDRT = homology-directed repair template, RNP = ribonucleoprotein, CLTA = Clathrin, Treg = regulatory T cells, HSC = hematopoietic stem cell, NK cells = natural killer cells, γδ T cells = gamma delta T cells.

Extended Data Fig. 7 Evaluation of small molecule inhibitor cocktails in primary human T cells.

(a) Evaluation of relative increase in percent knock-in using an ssDNA CD5-HA knock-in construct over varied concentrations of 5 different small molecule inhibitors assessed by flow cytometry. Red bars indicate concentrations chosen for subsequent experiments. (b) Comparison of relative percent knock-in (top), live cell counts (middle), and viability with Ghost Dye 780 (Tonbo) (bottom) with small molecule inhibitor combinations. Cocktails chosen for subsequent experiments are highlighted in red (M3814), blue (MT) and yellow (MTX). (c-d) Evaluation of Novobiocin effects on (c) live cell counts and (d) knock-in efficiency using a small CD5-HA ssDNA HDRT. (e) Evaluation of Novobiocin effects on knockin efficiency at varied concentration using a small CD5-HA ssDNA HDRT in combination with M3814, MT, and MTX inhibitors. Each experiment was performed with T cells from 2 independent healthy human blood donors represented by individual dots + mean. M = M3814, MT = M3814 + Trichostatin A, MTX = M3814 + Trichostatin A + XL413, NVB = Novobiocin.

Extended Data Fig. 8 Analysis of genome editing outcomes with CTS templates and small molecule inhibitors.

(a-c) Evaluation of genome editing outcomes by either (a) flow cytometry or (b) amplicon sequencing using small dsDNA, dsCTS, ssDNA, or ssCTS CD5-HA HDRTs at non-toxic concentrations (800 nM) with and without M, MT, and MTX inhibitor combinations. (c) Ratio of perfect:imperfect HDR events with each combination. (d) Comparison of dsCTS and ssCTS templates in combination with small molecular inhibitors in 5 different knock-in constructs using a large CD5-HA HDRT (-2.7 kb, n = 4 donors), a tNGFR knock-in to the IL2RA gene (~1.5 kb, n = 4 donors), an mCherry fusion in the clathrin gene (~1.5 kb, n = 4 donors), a near full length CTLA-4-GFP fusion to the CTLA4 gene (~2.1 kb, n = 6 donors), and a full length IL2RA-GFP fusion to the IL2RA gene (~2.3 kb, n = 6 donors). (e-f) Evaluation of (e) live cell counts and (f) viability + /- MT and MTX inhibitor combinations using 44 different knock-in constructs targeting a tNGFR marker across 22 different target loci with 2 gRNA per gene (g1 and g2). Panel a shows mean and individual values from two healthy blood donors. Panels b, c, e, and f show mean values from two healthy blood donors. Panel d shows mean + /- SD. CTS = Cas9 target site, HDRT = homology-directed repair template, dsCTS = dsDNA + CTS HDRT, ssCTS = ssDNA + CTS HDRT, M = M3814, MT = M3814 + TSA, MTX = M3814 + TSA + XL413.

Extended Data Fig. 9 IL2RA and CTLA4 ORF replacement strategies.

(a) Gating for GFP + cells are shown for WT and S166N IL2RA-GFP knock-in constructs. (b) Diagram of the CTLA4 gene (top), CTLA4 protein levels (bottom), and cutting efficiency (bottom) illustrating a screening panel of 12 gRNAs examined within exon 1 and intron 1. gRNAs were assessed in activated CD4 + T cells for protein disruption by CTLA4 flow cytometric analysis (flow plots and top row of numbers demonstrate the % of CTLA4-negative cells for each donor), and for cutting efficiency as determined by TIDE indel analysis51 (bottom row of numbers indicate the %indel at target locus). (c) CTLA4 expression levels assessed by flow cytometry with endogenous protein (black) and WT CTLA4-GFP knock-in protein (red) are shown for CD4- T cells, CD4 + T cells, and regulatory T cells with (dotted line) and without (solid line) stimulation. (d) Gating for GFPhi cells is shown for WT, R70W, R75W, and T124P CTLA4-GFP knock-in cells. Each experiment was performed with T cells from 2 independent healthy human blood donors. WT = Wild-Type, Treg = regulatory T cell.

Extended Data Fig. 10 Evaluation of a nonviral strategy for anti-BCMA CAR-T cell manufacturing.

(a-c) Comparison of (a) knockin efficiency (mean + /- SD), (b) flow cytometric immunophenotypes, and (c) tumor burden of MM1S-bearing NSG mice treated with TRAC anti-BCMA CAR-T cells generated using either AAV or non-viral ssCTS HDRTs (mean + /- SD). (d) Live cell counts for large-scale GMP-compatible manufacturing process at Day 7 and Day 10 post-activation. (e) Tumor burden (average radiance) of individual MM1S-bearing NSG mice treated with Unmodified T cells and TRAC anti-BCMA CAR-T cells generated in GMP-compatible anti-BCMA-CAR T cell scaleup experiment. (f) Kaplan–Meier analysis showing overall survival of MM1S xenotransplant NSG mice treated with anti-BCMA-CAR or unmodified T cells. (g-j) Targeted Locus Amplification (TLA) analysis for anti-BCMA TRAC CAR-T cell products generated in GMP-compatible scaleup experiments. (g) Integration site analysis based on TLA sequencing demonstrating targeted insertion at the expected TRAC locus on chromosome 14. (h) Mean percentage of perfect and imperfect HDR events by TLA sequencing from 2 independent healthy human blood donors. (i) Table of perfect HDR, imperfect HDR, and off-target events for individual donors by TLA sequencing. (j) TLA sequence coverage aligned on the TRAC anti-BCMA CAR ssCTS reference construct. Grey bars on Y axis indicate sequence coverage. Low coverage across the CTS indicates relatively rare non-HDR events incorporating the indicated bases. Panel a was performed with 3 independent healthy human blood donors. Open circles represent use of serum-free media post-electroporation, closed circles represent use of serum-containing media post-electroporation. Panel c performed with the indicated number of mice using T cell products generated from a matched single healthy blood donor. Panel d performed with 2 independent healthy human blood donors. Panel e-f performed with unmodified T cells (n = 4 mice) and BCMA-CAR T cells (n = 5 mice) generated from one healthy human blood donor. A second cohort of mice treated with cells from a second donor was excluded because tumor failed to efficiently engraft in control group. TLA Analyses performed in 2 independent healthy human blood donors. **P < 0.05; ns, not significant. P values obtained by (a) unpaired two tailed t-test, (c) two tailed Mann-Whitney test, or (f) log-rank Mantel–Cox test (survival). rAAV = recombinant adeno-associated virus, HDRT = homology-directed repair templates, RNP = ribonucleoprotein, TCR = T cell receptor, CTS = Cas9 target site, ssCTS = ssDNA + CTS HDRT, CAR = chimeric antigen receptor, GMP = good manufacturing practice, TLA = targeted locus amplification, LHA = left homology arm.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary.

Supplementary Table 1

Key reagents, calculations and statistics. ad, List of antibodies (a), gRNA sequences (b), primer sequences (c) and HDRT sequences used in this study (d). e, Knock-in efficiencies and fold-change for Fig. 3h. Primary immunodeficiency panel using small-molecule inhibitor combinations. Data are shown for two independent donors. f, Knock-in efficiencies and P values for Extended Data Fig. 8d. P values obtained by unpaired two-tailed t-test analysis. RC = reverse complement.

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Shy, B.R., Vykunta, V.S., Ha, A. et al. High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nat Biotechnol (2022). https://doi.org/10.1038/s41587-022-01418-8

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