Poor Cas9. If CRISPR were a new music trend, Cas9 would the band that was playing CRISPR long before it was cool, rode the CRISPR wave to stardom, alienated its fans with copyright battles, and is already under threat from the new kids.
Less metaphorically, Cas9 is an enzyme that uses RNA spacers (crRNAs) transcribed from CRISPR immunity arrays to chop up invading DNA that matches. Cas9 does all this as a single gene, making it much easier to work with than the multi-piece bands that make up other CRISPR systems. For a while, that made Cas9 nearly synonymous with CRISPR in synthetic biology, until another single-subunit effector burst onto the scene – Cpf1. Now it looks like these two are going to have to share the stage with even more RNA-guided endonucleases; a new paper just found 3 more subtypes.
Diversifying The Class 2 CRISPR Scene
Collectively, CRISPR systems with single-subunit effectors are known as Class 2. These are then subdivided even further into type II, fronted by Cas9, and type V, with Cpf1 headlining. First authors Sergey Shmakov and Omar Abudayyeh wanted to scout for more Class 2 talent, so they searched metagenomic databases for homologs of Cas1, the most conserved of the CRISPR proteins. Next, they looked for large proteins nearby, which could possibly be Class 2 effectors. This netted them 53 prospects, falling into two new subtypes of type V, and a new type VI system:
C2c1 (Class 2, candidate 1)
- Type V-B.
- Found on 18 genomes.
- The 2 tested loci were functional in E. coli.
- tracrRNAs (short RNAs that help separate the CRISPR array into individual spacers, or crRNAs) were required. As for Cas9, the tracrRNA could be fused to the crRNA to make a single short guide, or sgRNA.
- Targeted DNA with a 5’ PAM sequence TTN.
C2c3 (Class 2, candidate 3)
- Type V-C.
- The numbering is odd because it was the third candidate found, but it clusters with C2c1 and Cpf1 within type V.
- Only found in metagenomic sequences, so the actual species are unknown.
C2c2 (Class 2, candidate 2)
- Type VI – a new type!
- Found on 21 genomes.
- Very little homology to known sequences – possible RNase motifs?
- The 2 tested loci made mature crRNAs in E. coli; functionality was not reported.
- tracrRNAs were not expressed.
Diversification of the Class 2 CRISPR scene may or may not overshadow Cas9, but it should be great for fans. There looks to be a large variety of CRISPRs out there with different styles, including reliance on tracrRNAs, PAMs, nucleic acid preference (RNA vs DNA), and other innovations we haven’t even thought of yet. It may well be that no one system wins; maybe each will finds its own niche, or they may coexist and complement each other on playlists. Now if only they could figure out some better names and category nomenclature…
Don’t pretend you’re not a CRISPR groupie! Go check out the paper, from the labs of Feng Zhang and Eugene Koonin, at Molecular Cell, 2015.