A CRISPR-del-based pipeline for complete gene knockout in human diploid cells

Abstract

The advance of CRISPR/Cas9 technology has enabled us easily to generate gene knockout cell lines by introducing insertion/deletion mutations (indels) at the target site via the error-prone non-homologous end joining repair system. Frameshift-promoting indels can disrupt gene functions by generation of a premature stop codon. However, there is growing evidence that targeted genes are not always knocked-out by the indel- based gene disruption. In this study, we optimized CRISPR-del, which induces a large chromosomal deletion by cutting two different target sites, to perform complete gene knockout in non-transformed human diploid RPE1 cells. By improving several procedures, the optimized CRISPR-del allowed us to generate knockout cell lines harboring bi-allelic large chromosomal deletions in a high-throughput manner. Quantitative analyses show that the frequency of gene deletion with this approach is much higher than that of conventional CRISPR-del methods. The lengths of the deleted genomic regions demonstrated in this study are longer than those of 95% of the human protein-coding genes. Furthermore, the ability of this method to introduce a large chromosomal deletion enables the generation of a model cell line having a bi- allelic cancer-associated chromosomal deletion. Overall, these data lead us to propose that the optimized CRISPR-del is a high-throughput method for performing complete gene knockout in RPE1 cells.

ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

“…Guide RNA (sgRNA for Cas9 and crRNA for Cas12a) was transcribed in vitro from PCR-generated DNA templates according to a previous method (Komori et al, 2021). Briefly, for sgRNA, template DNA containing T7 promoter and sgRNA sequence was amplified by PCR from five different oligos.…”

“…Guide RNA (sgRNA for Cas9 and crRNA for Cas12a) was transcribed in vitro from PCR-generated DNA templates according to a previous method (Komori et al, 2021).…”