Quantitative analysis of CRISPR-del 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.