CRISPR/Cas9-mediated targeted knock-in of large constructs using nocodazole and RNase HII.

ABSTRACT: On-target integration of large cassettes via homology-directed repair (HDR) has several applications. However, the HDR-mediated targeted knock-in suffered from low efficiency. In this study, we made several large plasmids (12.1-13.4 kb) which included the CRISPR/Cas9 system along with a puromycin transgene as part of the large DNA donor (5.3-7.1 kb insertion cassettes) and used them to evaluate their targeted integration efficiency into a transgenic murine embryonic fibroblast (MEF) cell line carrying a single copy of a Venus transgene. We established a detection assay by which HDR events could be discriminated from the error-prone non-homologous end-joining (NHEJ) events. Improving the plasmid quality could considerably leverage the cell toxicity impediment of large plasmids. The use of t

SUBMITTER: Eghbalsaied S 

PROVIDER: S-EPMC9931768 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature