A group of researchers set out to characterize the genotypic abnormalities induced by Cas9 in human cells. To do this, they studied the tRNA gene. These researchers deleted two tRNA genes from the genomes of hyperploid human hepatocellular carcinoma (HepG2) and haploid chronic myeloid leukemia (HAP1) cells using the CRISPR/Cas9 system with dual gRNAs. The underlying alterations that caused rearrangement of the CRISPR/Cas9-targeted region were assessed by a customized de novo sequence assembly approach. Although a genomic region of interest was cleaved by Cas9 in this study, the cleaved fragment was duplicated, inverted, and inserted locally into the genomes of both HepG2 and HAP1 cells. The study also demonstrated the successful integration of exogenous DNA fragments into HepG2 cells.
Furthermore, the aberrant target-derived DNA fragments were shown to be still functional, tagged with active histones, and bound by RNA polymerase III. This highlights the fact that CRISPR/Cas9-based genomic engineering can lead to adverse effects on the target, and that inversion and duplication events can occur at the same time. In conclusion, this study reveals the complex genomic alterations that accompany CRISPR/Cas9 deletions.