One Anti-CRISPR to Rule Them All

Natural inhibitors of Cas9 are one of the most recent discoveries in the CRISPR world. A new paper in Cell looks at how one of the “Anti-CRISPRs” previously discovered inhibits a wide range of Cas9 orthologs.

As the CRISPR craze continues, one major lingering concern is Cas9 going rogue. Once we send Cas9 into a cell or an organism to cut DNA, how do we turn it off? Anti-CRISPRs, proteins that stop Cas9, have emerged as a new tool to take control of Cas9. These are small proteins from phages that stick to Cas9, and stop it from cutting DNA. Anti-CRISPRs are phages’ response to CRISPR, in the never-ending war between bacteria and their phages.

Last year and early this year, two groups discovered 5 different Anti-CRISPRs in phages of bacteria with CRISPR/Cas9 systems. The first three inhibited the Cas9 from Neisseria meningitidis (NmeCas9), and the latter two inhibited the widely used Cas9 from streptococcus pyogenes (SpyCas9). Both of these Cas9s have been used to edit mammalian genomes, but they are not the only ones.

Several other Cas9s from Campylobacter jejuni (CjeCas9) and Geobacillus stearothermophilus (GstCas9) have also been used to cut mammalian genomes. The new paper by Harrington and colleagues shows that one of the Anti-CRISPRs that inhibited NmeCas9, also inhibits CjeCas9 and GeoCas9 in vitro and in mammalian cells. That raises a fascinating question, how can one anti-CRISPR inhibit so many different Cas9s?

A good hypothesis is that the Anti-CRISPR is binding to a highly conserved region of Cas9, i.e. a region that is present in all of the Cas9s the Anti-CRISPR inhibits. The active site of the HNH domain of Cas9 is highly conserved, this is the part of Cas9 that cuts the DNA and would be an ideal spot for the Anti-CRISPR to target. The authors crystalized the HNH domain of NmeCas9 and found that the Anti-CRISPR indeed binds to this part of Cas9.

This finding suggests that a wide range of Cas9s can be inhibited by this particular Anti-CRISPR. In addition to enabling us to take control of these Cas9s for genome editing, this study sheds light on the smart ways phages use to combat CRISPR immunity.

 

 

Anti-CRISPR comic by Megan Hochstrasser of IGI