Though CRISPR initially encountered fame as a revolutionary method used to edit DNA, it can also be used to cleave strands of RNA. Interestingly, this technique was developed by the team at the Broad Institute that were the first to use CRISPR-Cas9 in mammalian cells. Here, instead of using the Cas9 endonuclease to cleave the nucleic acid, C2c2 is used. Like in the original CRISPR-Cas9 system, C2c2 is guided by a crRNA complementary to the desired sequence (except, of course, the sequence is now encoded by RNA rather than DNA). The C2c2 enzyme has two Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) domains, RNases that mediate cleave of ssRNA (Abudayyeh et al., 2016).
There are two distinct classes of CRISPR systems, delineated by the means of nucleic acid cleavage. Whilst Class I systems use multi-subunit protein complexes, Class II systems use single effector proteins. The original CRISPR-Cas9 system used in genome editing was originally identified in Streptococcus pyogenes; the C2c2 system is an example of a Class II Type VI-A CRISPR system isolated from Leptotrichia shahii (Abydayyeg et al., 2016). The discovery is encouraging: given what is known about the diversity of CRISPR loci across taxa, it is likely that more will have potential use in laboratories. C2c2 is specific to prokaryotic RNA, though shortly before its announcement, researchers at the University of California reported that they had manipulated CRISPR-Cas9 to act on mammalian RNA (Nelles et al., 2016).
Importantly, as it is a Class II system, C2c2 uses a single effector protein: when directed by the guide RNA, no other protein or complex of proteins is needed to mediate cleavage. Though not the first CRISPR system described that targets RNA (both the type III-A and –B systems cleave RNA), C2c2 is the first that uses just a single protein to do so. This is an important point, as inefficient delivery mechanisms have been a long-standing hurdle for gene therapies (Hill et al., 2016). Simple, smaller complexes are more easily transported to target cells.
Though researchers currently use RNA interference (RNAi) to post-transcriptionally silence genes, the C2c2 method may prove a more powerful tool. RNAi is a natural biological mechanism shared by many eukaryotic cells that involves the production of short interfering RNA (siRNA) or microRNA (miRNA) complementary to a target strand. Upon pairing with the target strand, the now-double stranded RNA (dsRNA) is cleaved by the RNA-induced silencing complex (RISC) (Castel and Martienssen, 2013). As a tool, it has been instrumental in genetics for allowing detailed study of eukaryotic cells, though it was not until recent years – namely, the advent of CRISPR and related technologies – that there was a prokaryotic equivalent.
Editing RNA has several advantages over editing DNA, largely because any edits introduced are impermanent. Thus, any mistakes or undesired effects are unlikely to have severe long-term consequences and the system can be modified by researchers to fix the problem.
Additionally, when editing DNA, unless very specific and often unknown changes are introduced into a regulatory region, it is difficult for researchers to control the degree to which gene expression is changed. It may be switched on or switched off, but any finer level of control is hard to achieve without more extensive knowledge of the gene. However, editing mRNA facilitates more precise control of gene expression, as it is easier to add new units to the strand and examine its effect on function (Broad Institute, 2016). For example, by adding small fluorescent tags to the RNA, researchers may gain insight into post-translational trafficking of mRNA.
There are also fewer ethical concerns associated with editing RNA, as the changes can never be transferred to the next generation (as is the case with germline engineering).
CRISPR-C2c2 is still a developing technology, though it holds great promise in both research and clinical settings. On the bench, it will act as a more powerful version of RNAi. Regarding its therapeutic potential, it may be used to target disease-causing RNAs produced by the cell or even RNA viruses such as HIV. Ultimately, development of the CRISPR-C2c2 system heralds a new means of studying RNA and its critical role in cellular function, paving the way for new disease treatments.
Abudayyeh, O.O., Gootenberg, J.S., Konermann, S., Joung, J., Slaymaker, I.M., Cox, D.B.T., Shmakov, S., Makarova, K.S., Semenova, E., Minakhin, L., Severinov, K., Regev, A., Lander, E.S., Koonin, E.V. and Zhang, F. (2016). C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573
Broad Institute (2nd June 2016). New CRISPR system for targeting RNA
Castel, S.E. and Martienssen, R.A. (2013). RNA interference in the nucleus: roles for small RNAs in transcription, epigenetics and beyond. Nature Reviews Genetics 14: 100-112
Hill, A.B., Chen, M., Chen, C.-K., Pfeifer, B.A. and Jones, C.H. (2016). Overcoming gene-delivery hurdles: physiological considerations for nonviral vectors. Trends in Biotechnology 34: 91-105
Nelles, D.A., Fang, M.Y., O’Connell, M.R., Xu, J.L., Markmiller, S.J., Dounda, J. A. and Yeo, G.W. (2016). Programmable RNA tracking in live cells with CRISPR/Cas9. Cell 165:488-496