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

ABSTRACT: Enhancing 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 2–3-fold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional 2–3-fold 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 non-viral chimeric antigen receptor (CAR)-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.

ORGANISM(S): Homo sapiens

PROVIDER: GSE202596 | GEO | 2022/05/12

REPOSITORIES: GEO

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

Project description:Enhancing 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 2–3-fold relative to dsDNA CTSs. Using small-molecule combinations that enhance HDR, we could further increase knock-in efficiencies by an additional 2–3-fold 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 non-viral chimeric antigen receptor (CAR)-T cell manufacturing, with knock-in efficiencies (46–62%) and yields (>1.5×109 modified cells) exceeding those of conventional approaches.

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