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A recent study by researchers from Sweden and the United Kingdom shows that CRISPR/Cas9-based genomic engineering can prompt unwanted on-target effects, and highlights the complexity of human DNA repair mechanisms in the presence of the powerful prokaryotic Cas9 nuclease. The paper is currently available on the bioRxiv* preprint server. 
sofia carlos's curator insight,
April 10, 2022 8:44 PM
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BigField GEG Tech's insight:
This review presents the mechanisms of different gene editing strategies and describes each of the common nuclease-based platforms, including zinc finger nucleases, TALE nucleases, meganucleases, and the CRISPR/Cas9 system. The authors summarize the progress made in applying genome editing to various areas of gene and cell therapy, including antiviral strategies, immunotherapies, and the treatment of monogenic hereditary disorders.
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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.