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Massively parallel knock-in engineering of human T cells

Nature Biotechnology volume 41, pages 1239–1255 (2023)Cite this article

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Abstract

The efficiency of targeted knock-in for cell therapeutic applications is generally low, and the scale is limited. In this study, we developed CLASH, a system that enables high-efficiency, high-throughput knock-in engineering. In CLASH, Cas12a/Cpf1 mRNA combined with pooled adeno-associated viruses mediate simultaneous gene editing and precise transgene knock-in using massively parallel homology-directed repair, thereby producing a pool of stably integrated mutant variants each with targeted gene editing. We applied this technology in primary human T cells and performed time-coursed CLASH experiments in blood cancer and solid tumor models using CD3, CD8 and CD4 T cells, enabling pooled generation and unbiased selection of favorable CAR-T variants. Emerging from CLASH experiments, a unique CRISPR RNA (crRNA) generates an exon3 skip mutant of PRDM1 in CAR-Ts, which leads to increased proliferation, stem-like properties, central memory and longevity in these cells, resulting in higher efficacy in vivo across multiple cancer models, including a solid tumor model. The versatility of CLASH makes it broadly applicable to diverse cellular and therapeutic engineering applications.

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Fig. 1: Establishment of CLASH system for massively parallel knock-in engineering.
Fig. 2: Timecourse in vitro CD8 and CD4 CLASH experiments identified genetic mutations that promote CAR-T cell persistence in long-term co-culture.
Fig. 3: In vivo CLASH in a mouse model of cancer with adoptive transfer of CAR-T pool.
Fig. 4: Individual functional analysis of immune genes scored in CLASH.
Fig. 5: PRDM1 Δexon3 CAR-Ts showed enhanced in vivo therapeutic efficacy in multiple mouse models of cancer.
Fig. 6: Molecular characterization of PRDM1 Δexon3 mutant CAR-T cells and genome-wide AAV integration.

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Data availability

All data generated or analyzed during this study are included in this article and its supplementary information files. Specifically, source data and statistics for non-high-throughput experiments, such as flow cytometry, qPCR, protein experiments and other molecular or cellular assays, are provided in an Excel file of source data and statistics. Processed data for genomic sequencing (for example, CLASH, RNA-seq, amplicon sequencing, MIPS and AAV off-target) and other forms of high-throughput experiments are provided as processed quantifications in Supplementary Datasets. Genomic sequencing raw data are being deposited to the Gene Expression Omnibus (GEO), with the following accession numbers: GSE207143 for all CLASH screens; GSE219061 for MIPS, Nextera and AAV integration off-target; GSE207404 for RNA-seq; and GSE201997 for CUT&RUN107,108,109,110. CLASH vectors and libraries are available via material transfer agreements. All other data and materials that support the findings of this research are available either via public repositories or from the corresponding author upon reasonable request to the academic community. Source data are provided with this paper.

Code availability

Analytic codes used to generate figures that support the findings of this study will be available from the corresponding author upon reasonable request.

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Acknowledgements

We thank M. Sznol, A. Bersenev, S. Seropian, I. Isufi, M. Müschen and D. Krause for discussions. We thank all members of the Chen laboratory as well as various colleagues in Yale Genetics, SBI, CSBC, MCGD, Immunobiology, BBS, YCC, YSCC and CBDS for assistance and/or discussions. We thank various Yale Core Facilities, such as YCGA, HPC, WCAC and KBRL, for technical support. S.C. is supported by NIH/NCI/NIDA (DP2CA238295, R01CA231112, U54CA209992-8697, R33CA225498 and 1RF1DA048811); DoD (W81XWH-20-1-0072 and W81XWH-21-1-0514); the Alliance for Cancer Gene Therapy; the Sontag Foundation (Distinguished Scientist Award); Pershing Square Sohn Cancer Research Alliance; Dexter Lu; the Ludwig Family Foundation; the Blavatnik Family Foundation; and the Chenevert Family Foundation. X.D. is supported by the Charles H. Revson Senior Postdoctoral Fellowship. J.J.P. is supported by an NIH Medical Scientist Training Program grant (T32GM136651). R.C. is supported by an NIH Medical Scientist Training Program grant (T32GM136651) and the National Research Service Award fellowship (F30CA250249). P.A.R. is supported by an NIH training grant (T32GM007499), the Lo Fellowship and NIH/NCI Diversity Supplement. S.S. is supported by a Mark Foundation for Cancer Research Emerging Leader Award, a Paul G. Allen Frontiers Group Distinguished Investigator Award and NIH/NIGMS R01GM122984.

Author information

Author notes

  1. Yaying Du

    Present address: Department of Thyroid and Breast Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

  2. Cun Liao

    Present address: Department of Colorectal and Anal Surgery, The First Affiliated Hospital of Guangxi Medical University, Nanning, China

  3. These authors contributed equally: Xiaoyun Dai, Jonathan J. Park, Yaying Du, Zhenkun Na, Stanley Z. Lam.

Authors and Affiliations

  1. Department of Genetics, Yale University School of Medicine, New Haven, CT, USA

    Xiaoyun Dai, Jonathan J. Park, Yaying Du, Stanley Z. Lam, Ryan D. Chow, Paul A. Renauer, Shan Xin, Zhiyuan Chu, Cun Liao, Paul Clark, Hongyu Zhao & Sidi Chen

  2. System Biology Institute, Yale University, West Haven, CT, USA

    Xiaoyun Dai, Jonathan J. Park, Yaying Du, Stanley Z. Lam, Ryan D. Chow, Paul A. Renauer, Shan Xin, Zhiyuan Chu, Cun Liao, Paul Clark & Sidi Chen

  3. Center for Cancer Systems Biology, Yale University, West Haven, CT, USA

    Xiaoyun Dai, Jonathan J. Park, Yaying Du, Stanley Z. Lam, Ryan D. Chow, Paul A. Renauer, Shan Xin, Zhiyuan Chu, Cun Liao, Paul Clark & Sidi Chen

  4. M.D.-Ph.D. Program, Yale University, West Haven, CT, USA

    Jonathan J. Park, Ryan D. Chow & Sidi Chen

  5. Molecular Cell Biology, Genetics, and Development Program, Yale University, New Haven, CT, USA

    Jonathan J. Park, Ryan D. Chow, Paul A. Renauer & Sidi Chen

  6. Department of Chemistry, Yale University, New Haven, CT, USA

    Zhenkun Na & Sarah Slavoff

  7. Institute of Biomolecular Design and Discovery, Yale University, West Haven, CT, USA

    Zhenkun Na & Sarah Slavoff

  8. Department of Biostatistics, Yale University School of Public Health, New Haven, CT, USA

    Jianlei Gu & Hongyu Zhao

  9. Immunobiology Program, Yale University, New Haven, CT, USA

    Zhiyuan Chu & Sidi Chen

  10. Computational Biology and Bioinformatics Program, Yale University, New Haven, CT, USA

    Hongyu Zhao

  11. Yale Center for Biomedical Data Science, Yale University School of Medicine, New Haven, CT, USA

    Hongyu Zhao & Sidi Chen

  12. Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, USA

    Sarah Slavoff

  13. Combined Program in the Biological and Biomedical Sciences, Yale University, New Haven, CT, USA

    Sidi Chen

  14. Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  15. Yale Comprehensive Cancer Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  16. Yale Stem Cell Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  17. Yale Liver Center, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

  18. Yale Center for RNA Science and Medicine, Yale University School of Medicine, New Haven, CT, USA

    Sidi Chen

Authors
  1. Xiaoyun Dai
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Contributions

Conceptualization: X.D. and S.C. Library design: J.J.P. and S.C. Experiment lead: X.D., Y.D. and Z.N. Analytic lead: J.J.P. and S.L. Additional data analysis: R.C., P.A.R. and J.G. Additional experiment support: S.X., Z.C., C.L. and P.C. Manuscript preparation: X.D., J.J.P., S.L., Z.N., Y.D. and S.C. Supervision: S.C., S.S. and H.Z. Research funding: S.C., S.S. and H.Z. Overall organization: S.C. and X.D.

Corresponding author

Correspondence to Sidi Chen.

Ethics declarations

Competing interests

A patent application has been filed by Yale University on CLASH (S.C., X.D., J.J.P. and Y.D. as inventors). S.C. is a founder of Cellinfinity Bio, which licensed the CLASH patent. S.C. is also a founder of EvolveImmune Tx, Chen Consulting and Chen Tech, unrelated to this study.

Peer review

Peer review information

Nature Biotechnology thanks Li Tang, Hyongbum Henry Kim and Joseph Fraietta for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–13 of supplementary datasets and tables

Reporting Summary

Supplementary Data

This zip file contains six datasets. (1) CLASH library, in vitro and in vivo screening experiments and analyses. (2) All CLASH-MIPS processed data and correlation analyses, with metadata. (3) All Nextera amplicon sequencing indel variant frequencies, with metadata. (4) PRDM1 Δexon3 CD22 CAR-T timecourse mRNA-seq. (5) Genome-wide chromatin binding of PRDM1 WT and exon3 skip mutant via CUT&RUN in human CD22 CAR-T cells. (6) All genome-wide AAV on-target and off-target integration processed data, with metadata.

Supplementary Table

Oligo sequences used in this study are listed in an Excel file.

Source data

Source Data

Statistical source data for Figs. 1 and 3–6 and Extended Data Figs. 1, 5–9 and 12.

Source Data Fig. 4

Unprocessed western blots

Source Data Fig. 6

Unprocessed western blots

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Dai, X., Park, J.J., Du, Y. et al. Massively parallel knock-in engineering of human T cells. Nat Biotechnol 41, 1239–1255 (2023). https://doi.org/10.1038/s41587-022-01639-x

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