Targeted sequencing of tp53 gene after CRISPR-mediated point mutation knock-in

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. The risk of off-target knock-ins was identified and we developed a workflow to ensure detection of correct knock-ins.