Researchers Identify ‘Anti-CRISPR’ Molecules that Inhibit Cas9 Enzyme Activity

Researchers Identify ‘Anti-CRISPR’ Molecules that Inhibit Cas9 Enzyme Activity

One of the problems with the use of the CRISPR gene editing tool for treating genetic diseases in humans is how to control gene editing precisely once CRISPR-Cas9 has been introduced. While CRISPR can be used to make accurate edits to DNA to replace or alter the functions of genes, off target effects are possible. Controlling the edits and reducing the potential for off-target effects remains a major challenge.

CRISPR was developed by bacteria as a mechanism for eliminating DNA introduced by viruses. Bacteria have also developed anti-CRISPR systems which disrupt the activity of the Cas nucleases which are responsible for binding to and cutting the DNA. By controlling Cas enzyme activity, bacteria have precise control over the DNA edits that are performed. Harnessing these anti-CRISPR mechanisms could help to make CRISPR gene editing much safer for humans.

A team of researchers has developed two high-throughput assays that have allowed them to identify small molecule inhibitors of Streptococcus pyogenes Cas9 (SpCas9) binding and cutting activity. The first assay allowed them to screed a wide range of different small molecules to assess the interactions between SpCas9 and a fluorophore-labeled segment of DNA. The second assay measured fluorescence changes which signaled cleavage of DNA in the reporter gene.

The combination of both assays allowed the researchers to identify two compounds that disrupt binding and cleavage of DNA in mammalian cells. The molecules remain stable in human plasma and the level of inhibition is dose-dependent and reversable. Since the molecules are small, they can passively enter cells.

While several anti-CRISPR proteins have previously been discovered which could serve the same purpose they have many disadvantages. The proteins are large and cannot pass through cells, they can be attacked by proteases, trigger an immune response, and any inhibitory action they have is permanent. The small molecule Cas9 inhibitors eliminate all of these problems and can be easily synthesized in large numbers at low cost, with little variability between batches. While the research is still in the early stages, it appears that these molecules are highly specific.

These small molecules therefore have considerable potential for use alongside SpCas9 gene editing technologies and could allow far greater control of gene, base, and epigenetic editing in CRISPR treatments for patients.

“These results lay the foundation for precise chemical control over CRISPR-Cas9 activities, enabling the safe use of such technologies,” explained co-author, Amit Choudhary, PhD. However, while there is potential for use in CRISPR-Cas9 gene editing applications in humans, further research is required as they are not yet ready for application in humans and they have only been tested for efficacy in the lab, not in organisms.

The next step for the researchers is to identify the exact binding sites and the mechanisms used for inhibiting Cas9 activity. Then the researchers can work on increasing their potency and determining whether they have any other effects in mammalian cells.

The study – A High-Throughput Platform to Identify Small-Molecule Inhibitors of CRISPR-Cas9 – was recently published in the journal Cell. DOI: 10.1016/j.cell.2019.04.009

Leave a Reply