|ZFIN ID: ZDB-PUB-180616-9|
Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9
Prykhozhij, S.V., Fuller, C., Steele, S.L., Veinotte, C.J., Razaghi, B., Robitaille, J.M., McMaster, C.R., Shlien, A., Malkin, D., Berman, J.N.
|Source:||Nucleic acids research 46(17): e102 (Journal)|
|Registered Authors:||Berman, Jason, Prykhozhij, Sergey, Razaghi, Babak, Veinotte, Chansey|
|PubMed:||29905858 Full text @ Nucleic Acids Res.|
Prykhozhij, S.V., Fuller, C., Steele, S.L., Veinotte, C.J., Razaghi, B., Robitaille, J.M., McMaster, C.R., Shlien, A., Malkin, D., Berman, J.N. (2018) Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9. Nucleic acids research. 46(17):e102.
ABSTRACTWe 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.