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.
- Nucleic acids research 46(17): e102 (Journal)
- Registered Authors
- Berman, Jason, Prykhozhij, Sergey, Razaghi, Babak, Veinotte, Chansey
- MeSH Terms
- Animals, Genetically Modified
- CRISPR-Cas Systems*
- Clustered Regularly Interspaced Short Palindromic Repeats
- Embryo, Nonmammalian
- Gene Editing/methods*
- Gene Knock-In Techniques/methods*
- Mutagenesis, Site-Directed/methods
- Point Mutation/genetics*
- 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.
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.
Genes / Markers
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Engineered Foreign Genes