Vectorology - GEG Tech top picks

Although CRISPR/Cas9 mainly generates short insertions or deletions at the target site, it can also make large deletions of DNA around the specific target site. These large deletions pose safety concerns and can reduce the efficiency of functional editing. The WFIRM team is looking at ways to reduce the risk of this happening. The research described in their recent paper, published recently in Nucleic Acids Research, aimed to combat the generation of unpredictable long DNA deletions on target and find a way to prevent this, a key step towards the development of gene editing therapies to treat genetic diseases. The team evaluated a variety of human cells and genes of interest and found that fusing DNA polymerase I or the Klenow fragment to the Cas9 enzyme minimised large, unanticipated deletions of genomic DNA without sacrificing the efficiency of genome editing. On the contrary, it even increased editing efficiency in primary human cells.  

CRISPR-Cas9 is a powerful genome editing technology, yet with off-target effects. Truncated sgRNAs (17nt) have been found to decrease off-target cleavage without affecting on-target disruption in 293T cells. In this work, the authors showed that both 17nt and 20nt sgRNAs expressed by lentiviral vectors induce ~95% knockout (KO) in 293T cells, whereas the KO efficiencies are significantly lower in iPSCs (60–70%) and MSCs (65–75%). Furthermore, they observed a decrease of 10–20 percentage points in KO efficiency with 17nt sgRNAs compared to full-length sgRNAs in both iPSCs and MSCs. These results indicate the importance of balancing on-target gene cleavage potency with off-target effects: when efficacy is a major concern such as genome editing in stem cells, the use of 20nt sgRNAs is preferable.

In a recent study, a team of researchers used a CRISPR/Cas9 system to evaluate the use of tRNA by deleting two tRNA genes from the genomes of hyperhepatocellular carcinoma and human chronic myeloid quasi-haploid leukaemia cells. The authors discovered numerous unexpected genomic changes at the target region using an improved droplet-based target enrichment approach followed by Oxford Nanopore Technology long-read sequencing. The method used in this study demonstrates that CRISPR/Cas9 can lead to the integration of endogenous and exogenous DNA fragments and also produce local inversions, duplications and insertions of functional target-derived fragments. This research presents evidence that a combination of duplication and inversion, as well as integration of exogenous DNA fragments and clustered interchromosomal rearrangements, can occur simultaneously. Furthermore, it was shown for the first time that the target-derived fragments were nevertheless functional despite these modifications, which may complicate mechanistic explanations. These results reveal a new example of unintended CRISPR/Cas9 editing events that can go unnoticed and have a significant impact on the conclusions drawn from experimental reads.