The ultra-precise version of the CRISPR genome editing tool is becoming more and more powerful. In recent days, researchers have used engineering enzymes to improve the accuracy of gene editing techniques to pinpoint DNA without introducing too many unnecessary mutations. The enzymes, published in the latest issue of Nature-Biotechnology, could make a method called base editing more viable and become a more effective tool for treating genetic diseases, Nature reported.
Base editing has better control than traditional CRISPR-Cas9 systems. Photo credit: Carlos Clarivan Science Photo Library
“The era of human genome editing is now at a fragile beginning, and we all have a responsibility to do everything possible to reduce adverse effects. “Especially when these techniques are beginning to enter clinical trials,” said David Liu, a chemical biologist at the Broad Institute in the United States, who led the study. “
In CRISPR-Cas9 editing, the researchers used Cas9 enzymes to cut the DNA double strands and then relied on the cell’s DNA repair mechanism to introduce changes in DNA sequences at that location. This method is effective, but has little control over what sequence series is introduced. In base editing, however, the ability of cas9 enzymes to cut DNA is destroyed and fused with other enzymes that chemically convert one “letter” of DNA into another. Cas9 directs these enzymes to the target position. There, instead of destroying two chains of DNA, they rewrite a particular “letter.”
But the technology still needs to be optimized. Last year, researchers reported that enzymes used to convert DNA base C into T act not only on targets specified by researchers, but also on other random locations in the genome. These off-target effects raise concerns about the use of the technology for gene therapy, as unnecessary DNA editing can cause potential harm.
What’s more, these changes are randomly scattered across the genome, and the only way to detect them is to sequence all the edited genomes — cumbersome and expensive.
To solve this problem, the team developed several ways to look for off-target mutations in bacteria and human cells without the need to sequence the entire genome. In one of these methods, the researchers inserted the base editor into bacteria and tested the microbial resistance to antibiotics. The more resistant bacterial cells are, the more active the DNA mutations in the base editor.
The team screened a variety of enzymes, including naturally occurring and synthetic enzymes, in search of higher-fidelity base editing enzymes. As a result, they found a group of enzymes that convert C into T without causing many off-target mutations.
Gao Caixia, a plant biologist at the Institute of Genetics and Developmental Biology at the Chinese Academy of Sciences, said reducing off-targeting is important for using base editing to treat diseases. She says the screening method is more exciting than the new enzyme itself. Because some base editors don’t work well in plant cells, she hopes to screen out effective tools.
In addition, in another paper in Nature-Biotechnology, researchers further developed Cas9 enzymes, which can target previously inaccessible areas of DNA.
Related papers: https://doi.org/10.1038/s41587-020-0414-6 (2020)