In the CRISPR hype up to now, DNA has gotten a raw deal. In all known CRISPR systems, RNA guides gang up with protein effectors (e.g., Cas9 and Cpf1) to chop up poor, abused DNA helices. Now, a new CRISPR protein – C2c2 – is giving DNA a reprieve, turning the tables on RNA instead.
Some CRISPR systems do cut RNA as well as DNA, but none are naturally RNA-specific. Recently, Feng Zhang’s lab at MIT found several new single-protein CRISPR effectors. Among them was the enigmatic C2c2, which looked like it might be an RNase. Since then, the lab has probed the workings of C2c2 from Leptotrichia shahii, finding that it does indeed target single-stranded (ss)RNA. Here’s the lowdown:
- C2c2 is a Class 2 CRISPR system (meaning it has a single-protein effector) and the founding member of Type VI.
- C2c2 has no DNase domains, but it does have two HEPN RNase domains, both of which are required for nuclease activity.
- With appropriate guide RNAs, C2c2 gives E. coli immunity to the MS2 ssRNA phage.
- Targeting C2c2 to mRNA prevents gene expression.
- A, C, and U are the preferred nucleotides just 3’ of the protospacer in target RNA.
- Single base-pair mismatches between the guide and target have little effect. The central region is most sensitive to double mismatches, and three consecutive mismatches anywhere prevent cleavage.
- C2c2 cleaves at uracil (U) bases outside of its binding site. Regions lacking secondary structure seem to be most vulnerable.
- When C2c2 is activated by binding to its target, it appears to rampage uncontrollably, chopping up so much collateral mRNA that it slows down growth of its host cell.
- Nuclease dead C2c2 binds its target with a strong KD of 7 nM.
This RNA-targeting CRISPR opens up a whole new realm of possibilities, and the authors suggest a few applications:
- Knocking down expression of a particular gene.
- Using the promiscuous activity of target-bound C2c2 to arrest cell growth.
- Using nuclease-dead C2c2 to tag mRNA, co-localize it with another molecule, or localize it to a particular compartment.
Any wagers on how long it’ll take to tame C2c2’s off-target RNase activity? We have a very nice highlighter on our desk that says under 2 years.
If you’re tired of beating on DNA, join first authors Omar Abudayyeh, Jonathan Gootenberg, and Silvana Konermann in giving RNA a few kicks at Science, June 2016.