| Autor: |
Shy, Brian R., Vykunta, Vivasvan S., Ha, Alvin, Talbot, Alexis, Roth, Theodore L., Nguyen, David N., Pfeifer, Wolfgang G., Chen, Yan Yi, Blaeschke, Franziska, Shifrut, Eric, Vedova, Shane, Mamedov, Murad R., Chung, Jing-Yi Jing, Li, Hong, Yu, Ruby, Wu, David, Wolf, Jeffrey, Martin, Thomas G., Castro, Carlos E., Ye, Lumeng |
| Zdroj: |
Nature Biotechnology; Apr2023, Vol. 41 Issue 4, p521-531, 11p |
| Abstrakt: |
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. Combinations of single-stranded DNA repair templates and small molecules markedly enhance genome editing. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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