‘Safeguard’ guide RNA regulates CRISPR-Cas9 activity and reduces unwanted mutations

Written by Kerstin Wright

Researchers from Kyushu University (Fukuoka City, Japan) and Nagoya University Graduate School of Medicine (Nagoya, Japan) have discovered a method of gaining greater control over CRISPR-Cas9-based gene editing, which may be the key to the effective treatment of genetic disorders with fewer unwanted side effects. 

Gene editing via CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, can target and modify specific DNA sequences. The Cas9 nuclease cuts DNA, guided by a synthetic guide RNA (gRNA), which recognizes the sequence of DNA that needs to be edited. Unwanted genes can therefore be deleted and new genes can be inserted where the Cas9 nuclease cuts the genome.

However, there are side effects associated with this technique. This includes edits to other genes, which were not the intended target. This typically occurs when off-target genes have a similar sequence to those being edited. Additionally, mutations can occur at the chromosome level, which has impeded the progression of gene therapy clinical trials for several indications. In an extreme case, this has caused deaths in the recipients of gene therapy for muscular dystrophy.

The researchers of the present study, which was published in Nature Biomedical Engineering, pinpointed this issue of high editing activity in CRISPR-based gene therapy, defining the issue as a lack of control over the Cas9 enzyme. They have put forward a strategy for restricting the activity of the enzyme to only edit the intended nucleotides of the faulty genes.

It was discovered that the addition of an extra cytosine extension to the 5’ end of the gRNA acted as a so-called ‘safeguard’, which gave the team greater control over the activity of the Cas9 enzyme. They termed this system ‘safeguard gRNA ([C]gRNA)’. This new technique improved the effectiveness of CRISPR-Cas9 gene editing and reduced a number of its unwanted side effects. It increased the efficiency of single-allele editing of the DNA as it can be edited on a single nucleotide-by-nucleotide basis, increased the efficiency of homology directed repair, reduced off-target effects and reduced cytotoxicity.

Masaki Kawamata, co-author of the paper, had this to say about its significance:

“In particular, we believe that this technology can make a significant contribution to the medical field. We are currently evaluating its therapeutic efficacy and safety for selected target diseases in cell and animal experiments and using it to help develop therapeutic drugs and gene therapy methods, especially for rare diseases for which no treatment methods have yet been established.”