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*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

58 Scopus citations


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 languageEnglish
Pages (from-to)521-531
Number of pages11
JournalNature Biotechnology
Issue number4
StatePublished - Apr 2023
Externally publishedYes


FundersFunder number
CIRM Alpha Stem Cell Clinic
Human Vaccines Project Michelson PrizeS10 RR028962
Mnemo Therapeutics
National Science Federation1933344, 1S10OD025096-01A1
UCSF Herbert Perkins Cellular Therapy and Transfusion Medicine
Weill Neurohub
National Institutes of HealthK08AI153767, L40AI140341
National Institute of Allergy and Infectious DiseasesP01AI138962, P01AI155393
National Institute of Diabetes and Digestive and Kidney DiseasesF30DK120213
Burroughs Wellcome Fund
Cancer Research Institute
Simons Foundation
California Institute for Regenerative MedicineINFR-10361
Larry L. Hillblom Foundation2020-D-002-NET
California Department of Fish and GameT32 DK007418, T32GM007618
National Center for Advancing Translational SciencesL30TR002983
Takeda Pharmaceuticals U.S.A.
University of California, San Francisco
Care-for-Rare Foundation
James B. Pendleton Charitable Trust
Innovative Genomics Institute
Parker Institute for Cancer Immunotherapy
Cytori Therapeutics
Deutsche Forschungsgemeinschaft


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