Departments of Pediatrics, Microbiology & Immunology, and Pathology, Dalhousie University, Halifax, NS, B3H 4R2, Canada.
Michael G. DeGroote School of Medicine, McMaster University,Hamilton, ON, L8S4L8, Canada.
Nucleic Acids Res. 2018 Sep 28;46(17):e102. doi: 10.1093/nar/gky512.
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.
我们使用成簇规律间隔短回文重复序列（CRISPR）/ Cas9 试剂和单链寡脱氧核苷酸将点突变基因敲入斑马鱼基因组的靶位点。通过等位基因特异性聚合酶链反应的新应用评估了基因敲入的效率，并通过高通量测序进行了验证。反义不对称寡核苷酸设计被发现是最成功的优化策略。然而，突变的切割位点与突变的接近程度以及硫代磷酸修饰的寡核苷酸也极大地提高了基因敲入的效率。我们通过开发一种正确的基因敲入检测工作流程，发现了一个以前未被认识到的脱靶转基因敲入的风险，并对此进行了规避。这些策略极大地促进了在斑马鱼中研究人类遗传疾病，并且对于增强其他动物模型系统中的基于 CRISPR 的方法也具有额外的适用性。
