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
N1 - Publisher Copyright:
© 2022, The Author(s), under exclusive licence to Springer Nature America, Inc.
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
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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 -