Department of Anatomy and Developmental Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-Ku, Hiroshima, 734-8553, Japan.
Department of Cellular and Molecular Anatomy and International Mass Imaging Center, Hamamatsu University School of Medicine, Hamamatsu, 431-3192, Japan.
Sci Rep. 2022 Jul 8;12(1):11681. doi: 10.1038/s41598-022-15776-5.
The introduction of small insertion/deletion (indel) mutations in the coding region of genes by the site-specific nucleases such as Cas9 allows researchers to obtain frameshift null mutants. Technically simple and costly reasonable genotyping methods are awaited to efficiently screen the frameshift null mutant candidates. Here, we developed a simple genotyping method called DST-PCR (Double-strand break Site-Targeted PCR) using "face-to-face" primers where the 3' ends of forward and reverse primers face each other at the position between 3-bp and 4-bp upstream of the PAM sequence, which is generally the Cas9-mediated double-strand break site. Generated amplicons are directly subjected to TBE-High-Resolution PAGE, which contains a high concentration of bis-acrylamide, for mutant clones detection with 1-bp resolution. We present actual cases of screening of CRISPR/Cas9-engineered knockout (KO) cells for six genes, where we screen indels to obtain potential KO cell clones utilizing our approach. This method allowed us to detect 1-bp to 2-bp insertion and 1-bp to 4-bp deletion in one or both alleles of mutant cell clones. In addition, this technique also allowed the identification of heterozygous and homozygous biallelic functional KO candidates. Thus, DST-PCR is a simple and fast method to screen KO candidates generated by the CRISPR/Cas9 system before the final selection of clones with sequencing.
通过 Cas9 等定点核酸酶在基因编码区引入小的插入/缺失（indel）突变，研究人员可以获得移码 null 突变体。目前需要技术简单且成本合理的基因分型方法来高效筛选移码 null 突变体候选者。在此，我们开发了一种称为 DST-PCR（双链断裂位点靶向 PCR）的简单基因分型方法，使用“面对面”引物，正向和反向引物的 3' 端在 PAM 序列上游 3-4 个碱基的位置彼此相对，该位置通常是 Cas9 介导的双链断裂位点。生成的扩增子直接进行 TBE-高分辨率 PAGE，其中包含高浓度的双丙烯酰胺，可实现 1 个碱基分辨率的突变克隆检测。我们介绍了使用 CRISPR/Cas9 工程化敲除（KO）细胞筛选六个基因的实际案例，我们通过该方法筛选 indel 以获得潜在的 KO 细胞克隆。该方法允许我们检测到一个或两个等位基因的突变细胞克隆中 1-2 个碱基的插入和 1-4 个碱基的缺失。此外，该技术还允许鉴定杂合和纯合双等位基因功能 KO 候选者。因此，DST-PCR 是一种在最终进行测序筛选克隆之前，筛选 CRISPR/Cas9 系统产生的 KO 候选者的简单、快速方法。
