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Precision Engineering of the Genome, Epigenome and Transcriptome | EK24


A new strategy for scarless homology-directed genome editing


Mar 8, 2021 12:00am ‐ Mar 8, 2021 12:00am

Description

A new strategy for scarless homology-directed genome editing Nina Reuven , Julia Adler , Nadav Myers, and Yosef Shaul Dept. of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel 76100 Targeted genome editing has been revolutionized by the implementation of CRISPR/Cas9 to specifically cleave genomic sequences. Yet, most templated changes demand homology-directed repair (HDR), active during the S/G2 phases of the cell cycle and thus only available in a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, simultaneously editing a gene of interest (GOI) and rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using repair of the ts mutation as a selectable marker, the selection is "scarless", since editing restores the wild type sequence. To test this strategy, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts together with a GOI resulted in up to 90% of the selected cells bearing the desired mutation in the GOI. This system gave efficient editing when using plasmid donors to insert large cassettes, and single-stranded oligonucleotide donors (ssODN) to effect smaller changes. Notable among the genes we edited was the proteasome subunit PSMB6, mutated T35A to eliminate its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating the utility of this system in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

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