Year: 2020
Type: article
Abstract: CRISPR/Cas9 has become a powerful tool for genome editing in zebrafish that permits the rapid generation of loss of function mutations and the knock-in of specific alleles using DNA templates and homology directed repair (HDR). We examined the efficiency of synthetic, chemically modified gRNAs and demonstrate induction of indels and large genomic deletions in combination with recombinant Cas9 protein. We developed an in vivo genetic assay to measure HDR efficiency and we utilized this assay to test the effect of altering template design on HDR. Utilizing synthetic gRNAs and linear dsDNA templates, we successfully performed knock-in of fluorophores at multiple genomic loci and demonstrate transmission through the germline at high efficiency. We demonstrate that synthetic HDR templates can be used to knock-in bacterial nitroreductase (ntr) to facilitate lineage ablation of specific cell types. Collectively, our data demonstrate the utility of combining synthetic gRNAs and dsDNA templates to perform homology directed repair and genome editing in vivo.
Source: Nucleic Acids Research
DOI: 10.1093/nar/gkaa085
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,992
Funders: American Liver Foundation, National Institutes of Health, Weill Cornell Medical College, National Institute on Alcohol Abuse and Alcoholism, National Cancer Institute, National Institute of General Medical Sciences, National Institute of Diabetes and Digestive and Kidney Diseases, National Center for Advancing Translational Sciences, Department of Surgery
Awards: G2050434022, G2768190413, Estrogen Regulation of Hepatic Growth, Understanding the role of Polycomb Repressive Complex 2 in melanoma, The role of mechanical signaling in alcoholic liver disease and hepatocellular carcinoma, G3376477779, Clinical and Translational Science Center, Metabolic Regulation of Liver Growth, G6503237232, Understanding the mechanism of H3K9 HMT mediated suppression of melanoma, G7016177623, A Community Zebrafish Resource for Modeling GWAS Biology, Weill Cornell/Rockefeller/Sloan Kettering MST Program, Clinical and Translational Science Center, J. NRSA Training Core
OpenAlex ID: W3008149374