Scientists have developed a new approach to improve the safety of the CRISPR-Cas9 gene editing system by controlling its activity. While the CRISPR-Cas9 system is efficient in genome editing, studies have revealed frequent off-target effects, p53 activation, and cytotoxicity with severe DNA damage, making it difficult to obtain knockout and homology-directed repair clones in human induced pluripotent stem cells.
In order to improve the system’s safety, various options such as anti-Cas9 proteins, small-molecule inhibitors, and oligonucleotides have been explored to limit Cas9 activity. Scientists have now developed a convenient and accurate experimental system, called allele-specific indel monitor system (AIMS), to visualise genome editing dynamics at the single-cell level in living cells.
They used this system to systematically analyse bi-allelic and mono-allelic editing and re-assess the sequence configuration of commonly used guide RNAs to determine if simple guide RNA modification could enable programmable Cas9 inhibition.
The team found that adding cytosine stretches to the 5′-end of conventional guide RNAs reduced genome editing efficiency in a length-dependent manner via multiple mechanisms. The study also established distinct optimal windows of Cas9 activity for different applications, including safe bi-allelic editing, mono-allelic editing, and HDR-based generation and correction of disease-associated single-nucleotide substitutions free from p53 activation.
Link to Research: https://www.nature.com/articles/s41551-023-01011-7
Published on April 11, 2023 and Last Updated on April 11, 2023 by: Priyank Pandey
