Jinhai Wang, Indira Medina Torres, Mei Shang, Jacob Al-Armanazi, Hamza Dilawar, Darshika U Hettiarachchi, Abel Paladines-Parrales, Barrett Chambers, Kate Pottle, Misha Soman, Baofeng Su, Rex A Dunham
CRISPR/Cas9-mediated multiplex genome editing (MGE) conventionally uses multiple single-guide RNAs (sgRNAs) for gene-targeted mutagenesis via the non-homologous end joining (NHEJ) pathway. MGE has been proven to be highly efficient for functional gene disruption/knockout (KO) at multiple loci in mammalian cells or organisms. However, in the absence of a DNA donor, this approach is limited to small indels without transgene integration. Here, we establish the linear double-stranded DNA (dsDNA) and double-cut plasmid (dcPlasmid) combination-assisted MGE in channel catfish (Ictalurus punctatus), allowing combinational deletion mutagenesis and transgene knock-in (KI) at multiple sites through NHEJ/homology-directed repair (HDR) pathway in parallel...
January 13, 2024: International Journal of Biological Macromolecules