Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Issue 17 (14th June 2018)

• Record Type: Journal Article
• Authors: Prykhozhij, Sergey V; Fuller, Charlotte; Steele, Shelby L; Veinotte, Chansey J; Razaghi, Babak; Robitaille, Johane M; McMaster, Christopher R; Shlien, Adam; Malkin, David; Berman, Jason N
• Is Part Of: Nucleic acids research. Volume 46:Issue 17(2018)
• Page Start: e102
• Page End: e102
• Publication Date: 2018-06-14
• Subjects: Nucleic acids -- Periodicals; Molecular biology -- Periodicals; 572.805
• DOI: 10.1093/nar/gky512
• Languages: English
• ISSNs: 0305-1048

Abstract

Abstract: We have optimized point mutation knock-ins into zebrafish genomic sites using clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 reagents and single-stranded oligodeoxynucleotides. The efficiency of knock-ins was assessed by a novel application of allele-specific polymerase chain reaction and confirmed by high-throughput sequencing. Anti-sense asymmetric oligo design was found to be the most successful optimization strategy. However, cut site proximity to the mutation and phosphorothioate oligo modifications also greatly improved knock-in efficiency. A previously unrecognized risk of off-target trans knock-ins was identified that we obviated through the development of a workflow for correct knock-in detection. Together these strategies greatly facilitate the study of human genetic diseases in zebrafish, with additional applicability to enhance CRISPR-based approaches in other animal model systems.