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.
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.
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.
Project description:The RNA-guided DNA endonuclease Cas9 has emerged as a powerful new tool for genome engineering. Cas9 creates targeted double-strand breaks (DSBs) in the genome. Knock-in of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient. Here, we report that linear DNAs (single and double-stranded) engage in a high-efficiency HDR mechanism that requires only ~35 nucleotides of homology with the targeted locus to introduce edits ranging from 1 to 1000 nucleotides. We demonstrate the utility of linear donors by introducing fluorescent protein tags in human cells and mouse embryos using PCR fragments. We find that repair is local, polarity-sensitive, and prone to template switching, characteristics that are consistent with gene conversion by synthesis-dependent strand-annealing (SDSA). Our findings enable rational design of synthetic donor DNAs for efficient genome editing.
