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 YeJonathan H. Esensten, Justin Eyquem, Alexander Marson

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

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

All Science Journal Classification (ASJC) codes

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

UN SDGs

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

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10.1038/s41587-022-01418-8

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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{9b3c66efb85441f7a6e88ec910a8a08f,

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 = "الإنجليزيّة",

volume = "41",

pages = "521--531",

journal = "Nature biotechnology",

issn = "1087-0156",

publisher = "Nature Research",

number = "4",

}

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 - http://www.scopus.com/inward/record.url?scp=85137064845&partnerID=8YFLogxK

U2 - 10.1038/s41587-022-01418-8

DO - 10.1038/s41587-022-01418-8

M3 - مقالة

C2 - 36008610

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

Abstract

All Science Journal Classification (ASJC) codes

UN SDGs

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