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.  

Here, the authors have ablated the CCR5 and CXCR4 genes in human CD4+ cell lines and primary CD4+ T cells, simultaneously, using CRISPR/Cas9. The efficiency of gene modification is as high as 55% for CCR5 and 36% for CXCR4 in CD4+ cell lines through transduction of a single lentiviral vector (LV-X4R5).  Moreover LV-X4R5 disrupted over 12% of CCR5 and 10% of CXCR4 in primary human CD4+ T cells, which were rendered resistant to HIV-1 NL4-3 and HIV-1 YU-2 in vitro.  Their results demonstrate the efficacy of CRISPR/Cas9 in multiplex gene modification on peripherally circulating CD4+ T cells, which may used to design functional cure for HIV-1 infection.

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 genome editing, it is crucial to ensure target-specific editing in diseased cells and to avoid altering the genome of spectator cells. Since a substantial fraction, i.e. 30-90%, of an intravenously injected dose of lipid nanoparticles (LPNs) accumulates in the liver, off-target editing in this organ must be minimized. According to a study published in Nature Biomedical Engineering , LNPs can be used to deliver two types of anti-CRISPR nucleic acid to the liver to disrupt genome editing in hepatocytes, thereby improving the specificity of CRISPR-Cas genome editing in the extrahepatic tissues and organs.  To determine whether anti-CRISPR oligonucleotides could be used to reduce off-target editing in the liver, researchers first treated mice stably expressing Cas9 tagged with GFP with anti-CRISPR or scrambled oligonucleotides formulated in NLPs that preferentially target the liver. Two hours later, they treated the same mice with the same NPLs, but instead encapsulated gRNA targeting the GFP locus. Compared to scrambled oligonucleotides, the anti-CRISPR ones reduced the frequency of deletion mutations in hepatocytes by more than twofold. Therefore, Cas9-mediated on-target genome editing in extrahepatic tissues could be maintained while limiting off-target editing in the liver.