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


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
JournalNature Biotechnology
StateAccepted/In press - 2022
Externally publishedYes


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