High-yield genome engineering in primary cells using a hybrid ssDNA repair template and small-molecule cocktails.
Brian R ShyVivasvan S VykuntaAlvin HaAlexis TalbotTheodore L RothDavid N NguyenWolfgang G PfeiferYan Yi ChenFranziska BlaeschkeEric ShifrutShane VedovaMurad R MamedovJing-Yi Jing ChungHong LiRuby YuDavid WuJeffrey WolfThomas G MartinCarlos E CastroLumeng YeJonathan H EsenstenJustin EyquemAlexander MarsonPublished in: Nature biotechnology (2022)
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 × 10 9 modified cells) exceeding those of conventional approaches.
Keyphrases
- small molecule
- induced apoptosis
- genome wide
- cell cycle arrest
- healthcare
- crispr cas
- oxidative stress
- primary care
- signaling pathway
- copy number
- circulating tumor
- rheumatoid arthritis
- endoplasmic reticulum stress
- cell death
- dna methylation
- gene expression
- single cell
- genome editing
- cell proliferation
- idiopathic pulmonary fibrosis
- high resolution
- circulating tumor cells
- bone marrow
- simultaneous determination
- genome wide identification
- interstitial lung disease
- candida albicans