The post-holiday party period is always an excellent time to reflect that there can always be too much of a good thing. Whether it’s cookies, champagne, or Bing Crosby, sometimes you just need an off-switch. The same can be true of Cas9 – leaving it turned on after your gene editing is done, or leaky expression in the wrong cell type or at the wrong time, can increase unwanted mutations. Cas9-piloted gene drives (on which the United Nations recently allowed research to continue), could also be made safer if Cas9 could be turned off at will.
We already have several strategies for activatable Cas9, but a rapid off switch was still missing from the gene-editing repertoire. However, CRISPR is a nuclear weapon, and where there are nuclear weapons, there is an arms race, even if the nuclei are cellular instead of atomic. In this case, the arms race is between CRISPR-armed bacteria and viruses trying to sneak past its defenses.
For Type I CRISPRs, which are more complex and thus less useful for human tinkerers, the arms race resulted in viruses evolving anti-CRISPR proteins as a missile defense shield to deactivate CRISPR systems before they can do their job. With that as inspiration, April Pawluk, working with Alan Davidson at the University of Toronto, reasoned that anti-CRISPRs effective against Cas9 (of the Type II CRISPR clan) likely exist somewhere in biology. After an extensive search through genetic sequences, here’s what she found:
- Invading DNA found in bacteria with Type II CRISPRs often has regulator genes similar to those found next to Type I anti-CRISPRs.
- Several genes next to putative Type II anti-CRISPR regulators work to shoot down the activity of Cas9 from Neisseria meningitidis (NmeCas9), including in human cells.
- The anti-CRISPRs also block Cas9 from binding DNA, so they could be useful for controlling the epigenome and regulating gene expression with nuclease dead dCas9.
- Type II anti-CRISPRs work against Cas9s closely related to their preferred targets; i.e., anti-CRIPSR targeting Brackiella oedipodis Cas9 also works against NmeCas9.
- However, anti-CRISPRs have an evolutionary distance limit – anti-CRISPRs from N. meningitidis and B. oedipodis are powerless against the most commonly used SpyCas9, and anti-CRISPRs cannot bridge the Type I-Type II divide.
- Whoever decorated the CRISPR phylogenetic tree did a pretty good job spreading out the anti-CRISPR homologs, so it seems likely there are many more anti-CRISPRs to target even more Cas9s hiding in the needles.
In addition to adding four Cas9 kill switches to our synthetic biology arsenal, this study is yet another example of the evolutionary Red Queen arms race, as well as showing how many awesome and useful things are still out there to discover.
Speaking of discovering more, before you hit the kill switch on your web browsing device, be sure to check out the paper from Cell, Dec. 2016.