A pair of primers facing at the double-strand break site enables to detect NHEJ-mediated indel mutations at a 1-bp resolution

Abstract

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.

Circadian oscillation in primary cilium length by clock genes regulates fibroblast cell migration

“…Detailed methods for the generation of knockout (KO) cell lines have been previously described (Phua et al , 2017; Ijaz & Ikegami, 2021; Ijaz et al , 2022). Bmal1‐KO, Kif3a‐KO, and Bbs2‐KO cells were generated using CRISPR/Cas9‐based genome‐editing techniques.…”

“…Single‐cell clones were generated by the limiting dilution culture method. Mutant cells were screened by double‐strand break site‐targeted PCR genotyping (Ijaz et al , 2022). KO cells were identified by DNA sequencing.…”

Circadian oscillation in primary cilium length by clock genes regulate fibroblast cell migration

“…Single cell clones were generated by the limiting dilution culture method. Mutant cells were screened by double-strand break site-targeted PCR genotyping (Ijaz et al ., 2022). KO cells were identified by DNA sequencing.…”

“…Detailed methods for the generation of Knock-out (KO) cell lines have been previously described (Ijaz & Ikegami, 2021; Ijaz et al , 2022; Phua et al ., 2017). Bmal1-KO, Kif3a-KO, and Bbs2-KO cells were generated using CRISPR/Cas9-based genome-editing techniques.…”