CRISPR-Cas9 technology is constantly evolving. Variants of Cas9 can be used for genome editing, activating gene expression, repressing gene expression, and much more. But there’s one thing that’s been missing: a way to shut off Cas9’s activity after it’s been turned on. The concern is that the longer Cas9 remains active in a cell, the greater chances there are for off-target edits to occur. Although methods to switch on Cas9 activity using light or drugs have been developed, the field lacked an “off-switch” for Cas9.
That is, until the recent discovery of anti-CRISPRs by the Sontheimer and Davidson labs. Anti-CRISPRs are phage-derived small protein inhibitors of CRISPR-Cas systems that help phages evade the CRISPR-Cas immune system of bacteria. Anti-CRISPRs were originally found in type I CRISPR-Cas systems. This wasn’t immediately useful to scientists since genome engineering largely relies on type II CRISPR-Cas systems, but recently Pawluk et al discovered three anti-CRISPRs for a type II system in Neisseria meningitidis (Nme). And while Streptococcus pyogenes Cas9 (SpyCas9) is the most used and well studied CRISPR system, N. meningitidis can also be used for genome editing. Check out Esvelt et al and Hou et al for more information on this.
Anti-CRISPRs Prevent Cas9 Activity
When these three Nme anti-CRISPRs were expressed in N. meningitidis, they inhibited NmeCas9 activity (see figure 1 of Pawluk et al). They also inhibited NmeCas9 when expressed in HEK293 cells. For these experiments, cells were transfected with three plasmids expressing the following: 1) NmeCas9, 2) a sgRNA, and 3) an anti-CRISPR (Acr). The expression of Acr reduced editing from ~30% in control samples to 0-10% in Acr samples, as measured via the T7 endonuclease 1 (T7E1) assay. See Figure 1 for a quick schematic of the cell culture experiments and summary of key T7E1 results.
Anti-CRISPRS Prevent dCas9 Tethering
Besides serving as “off-switches” for genome editing, one of the NmeCas9 anti-CRISPRs, AcrIIC3Nme, also blocked tethering of “dead” NmeCas9 to DNA. To test this, GFP-labeled dNmeCas9 and mCherry-labeled dSpyCas9 were expressed in U2OS cells along with their telomeric-targeting sgRNAs. When no anti-CRISPR is present, dNmeCas9 (green) and dSpyCas9 (red) co-localize (yellow) (figure 2A). However, expression of an anti-CRISPR eliminates GFP-dNmeCas9 loci (figure 2B), demonstrating its potent inhibition of dNmeCas9 DNA binding.
Anti-CRISPRs make yet another addition to the Cas9 genome editing toolbox. Pawluk et al demonstrate how they can inhibit Cas9 genome editing in bacterial and mammalian cells, and can also inhibit dCas9 DNA-binding in mammalian cells. It’s possible that yet to be discovered anti-CRISPRs allow dCas9 DNA binding but prevent catalytic activity. You can find the plasmids used in Pawluk et al here. Let us know about your experience with using anti-CRISPRs in the comments!
1. Pawluk, A., et al. Davidson, A. R. (2016). Naturally Occurring Off-Switches for CRISPR-Cas9. Cell,167(7). PMID: 27984730.
Additional Resources on the Addgene Blog
- First time CRISPR user? Check out this post.
- Learn how to Choose the Best Cas9 Variant for Your Next Experiment
- Read more about Optogenetic Control of CRISPR
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