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

Brian R. Shy; Vivasvan S. Vykunta; Alvin Ha; Alexis Talbot; Theodore L. Roth; David N. Nguyen; Wolfgang G. Pfeifer; Yan Yi Chen; Franziska Blaeschke; Eric Shifrut; Shane Vedova; Murad R. Mamedov; Jing Yi Jing Chung; Hong Li; Ruby Yu; David Wu; Jeffrey Wolf; Thomas G. Martin; Carlos E. Castro; Lumeng Ye; Jonathan H. Esensten; Justin Eyquem; Alexander Marson

doi:10.1038/s41587-022-01418-8

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

Brian R. Shy^*
, Vivasvan S. Vykunta
, Alvin Ha
, Alexis Talbot
, Theodore L. Roth
, David N. Nguyen
, Wolfgang G. Pfeifer
, Yan Yi Chen
, Franziska Blaeschke
, Eric Shifrut
, Shane Vedova
, Murad R. Mamedov
, Jing Yi Jing Chung
, Hong Li
, Ruby Yu
, David Wu
, Jeffrey Wolf
, Thomas G. Martin
, Carlos E. Castro
, Lumeng Ye

Jonathan H. Esensten, Justin Eyquem, Alexander Marson^*

^*Corresponding author for this work

University of California at San Francisco
Innovative Genomics Institute
Institut national de la santé et de la recherche médicale
Ohio State University
GenScript Biotech
Chan Zuckerberg Biohub

Research output: Contribution to journal › Article › peer-review

151 Scopus citations

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.

Original language	English
Pages (from-to)	521-531
Number of pages	11
Journal	Nature Biotechnology
Volume	41
Issue number	4
DOIs	https://doi.org/10.1038/s41587-022-01418-8
State	Published - Apr 2023
Externally published	Yes

Funding

Funders	Funder number
CIRM Alpha Stem Cell Clinic
Cytori Therapeutics
Takeda Pharmaceuticals U.S.A.
Cancer Research Institute
Parker Institute for Cancer Immunotherapy
Innovative Genomics Institute
Deutsche Forschungsgemeinschaft
University of California, San Francisco
Mnemo Therapeutics
UCSF Herbert Perkins Cellular Therapy and Transfusion Medicine
Burroughs Wellcome Fund
James B. Pendleton Charitable Trust
Simons Foundation
Weill Neurohub
Care-for-Rare Foundation
National Institutes of Health	K08AI153767, L40AI140341
National Science Foundation	1933344
Human Vaccines Project Michelson Prize	S10 RR028962
National Institute of Allergy and Infectious Diseases	P01AI138962, P01AI155393
National Center for Advancing Translational Sciences	L30TR002983
National Institute of Diabetes and Digestive and Kidney Diseases	F30DK120213
California Institute for Regenerative Medicine	INFR-10361
California Department of Fish and Game	T32 DK007418, T32GM007618
National Science Federation	1S10OD025096-01A1
Larry L. Hillblom Foundation	2020-D-002-NET

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 9 Industry, Innovation, and Infrastructure

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10.1038/s41587-022-01418-8

Cite this

Shy, B. R., Vykunta, V. S., Ha, A., Talbot, A., Roth, T. L., Nguyen, D. N., Pfeifer, W. G., Chen, Y. Y., Blaeschke, F., Shifrut, E., Vedova, S., Mamedov, M. R., Chung, J. Y. J., Li, H., Yu, R., Wu, D., Wolf, J., Martin, T. G., Castro, C. E., ... Marson, A. (2023). High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails. Nature Biotechnology, 41(4), 521-531. https://doi.org/10.1038/s41587-022-01418-8

@article{aeb6d5c7a6d14d22a74eca402a4b9a81,

title = "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.",

author = "Shy, \{Brian R.\} and Vykunta, \{Vivasvan S.\} and Alvin Ha and Alexis Talbot and Roth, \{Theodore L.\} and Nguyen, \{David N.\} and Pfeifer, \{Wolfgang G.\} and Chen, \{Yan Yi\} and Franziska Blaeschke and Eric Shifrut and Shane Vedova and Mamedov, \{Murad R.\} and Chung, \{Jing Yi Jing\} and Hong Li and Ruby Yu and David Wu and Jeffrey Wolf and Martin, \{Thomas G.\} and Castro, \{Carlos E.\} and Lumeng Ye and Esensten, \{Jonathan H.\} and Justin Eyquem and Alexander Marson",

note = "Publisher Copyright: {\textcopyright} 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.",

year = "2023",

month = apr,

doi = "10.1038/s41587-022-01418-8",

language = "אנגלית",

volume = "41",

pages = "521--531",

journal = "Nature Biotechnology",

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Shy, BR, Vykunta, VS, Ha, A, Talbot, A, Roth, TL, Nguyen, DN, Pfeifer, WG, Chen, YY, Blaeschke, F, Shifrut, E, Vedova, S, Mamedov, MR, Chung, JYJ, Li, H, Yu, R, Wu, D, Wolf, J, Martin, TG, Castro, CE, Ye, L, Esensten, JH, Eyquem, J & Marson, A 2023, 'High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails', Nature Biotechnology, vol. 41, no. 4, pp. 521-531. https://doi.org/10.1038/s41587-022-01418-8

TY - JOUR

T1 - High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails

AU - Shy, Brian R.

AU - Vykunta, Vivasvan S.

AU - Ha, Alvin

AU - Talbot, Alexis

AU - Roth, Theodore L.

AU - Nguyen, David N.

AU - Pfeifer, Wolfgang G.

AU - Chen, Yan Yi

AU - Blaeschke, Franziska

AU - Shifrut, Eric

AU - Vedova, Shane

AU - Mamedov, Murad R.

AU - Chung, Jing Yi Jing

AU - Li, Hong

AU - Yu, Ruby

AU - Wu, David

AU - Wolf, Jeffrey

AU - Martin, Thomas G.

AU - Castro, Carlos E.

AU - Ye, Lumeng

AU - Esensten, Jonathan H.

AU - Eyquem, Justin

AU - Marson, Alexander

PY - 2023/4

Y1 - 2023/4

N2 - 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.

AB - 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.

UR - https://www.scopus.com/pages/publications/85137064845

U2 - 10.1038/s41587-022-01418-8

DO - 10.1038/s41587-022-01418-8

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C2 - 36008610

AN - SCOPUS:85137064845

SN - 1087-0156

VL - 41

SP - 521

EP - 531

JO - Nature Biotechnology

JF - Nature Biotechnology

IS - 4

ER -

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

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UN SDGs

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