Targeted sequencing of tp53 gene after CRISPR-mediated point mutation knock-in. Danio rerio strain:Casper

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.

本研究利用成簇规律间隔短回文重复序列（CRISPR）/Cas9试剂与单链寡脱氧核苷酸，实现了斑马鱼基因组位点的点突变敲入优化。通过等位基因特异性聚合酶链式反应的创新应用评估敲入效率，并经高通量测序验证结果。研究发现，反义不对称寡核苷酸设计为最优的优化策略；此外，切割位点与突变位点的间距以及硫代磷酸寡核苷酸修饰，也可显著提升敲入效率。本研究还明确了脱靶敲入的风险，并开发了一套可确保正确敲入检测的工作流程。