Once a relatively obscure topic of research, clustered regularly interspaced short palindromic repeats (CRISPR) loci are one of the primary components of a prokaryotic adaptive immune system. Put simply, the defensive mechanism works by incorporating parts of an invading virus’ DNA into spacer sequence. If the invader then re-invades and is recognised, these spacers are transcribed into CRISPR-RNAs (crRNAs) and used to direct CRISPR-associated (cas) nucleases to the virus, resulting in degradation of the viral genome (Barrangou et al., 2007). However, once it was adapted for use as a genome-editing technology, interest in CRISPR-Cas9 dramatically increased.
Since scientists first co-opted CRISPR-Cas9 for use as a laboratory technique, many different versions have been developed. Each was designed to tackle a specific problem: originally just used to introduce simple insertions or deletions into genomes, derivations of the CRISPR-Cas9 can now be used to edit RNA, modulate gene activity, and introduce several changes to genomes simultaneously.
CRISPR interference (CRISPRi) is one such development. Using a catalytically “dead” Cas9 (dCas9), CRISPRi can be used to post-transcriptionally repress gene expression or to increase gene activity (sometimes termed CRISPR activation, or CRISPRa) (Shalem, 2015). Two amino acid substitutions (D10A and H840A) cause Cas9 to lose its enzymatic activity, meaning it is no longer able to cleave DNA. For a technique based upon its ability to cleave DNA with high specificity, rendering of the key components inactive initially seems counter-intuitive. However, the dCas9 can still be guided by the single-guide RNA (sgRNA) and sterically hinder gene expression by either repressing initiation of transcription or blocking elongation of an existing transcript (Larson et al., 2013). The dCas9 can achieve this steric hinderance alone, though to increase efficiency it can be complexed with a Krüppel-associated box (KRAB) domain (Gilbert, 2013).
With respect to its ability to knock-down gene activity, CRISPRi is strikingly similar to RNA interference (RNAi). Based on a normal cellular mechanism that uses short interfering RNA (siRNA) or short hairpin RNA (shRNA) complementary to the target mRNA, RNAi allowed researchers to modulate gene activity without completely losing expression of that gene. For genes that prove lethal when knocked-out or deleted, this is incredibly useful as it allows in-vivo study of its function. Even in other genes, lowering expression to a significant degree can allow for useful insights into gene-dosage effects. It has some clinical applications, too: in 2013, the results of a clinical trial involving forty-one patients cancer patients showed that RNAi was effective at targeting genes encoding VGEF and KSP, proteins commonly involved in tumour cell development. This lead to complete remission in some patients (Tabernero et al., 2013).
As CRISPR has already gained widespread popularity and support amongst the research community, some have predicted it will eclipse RNAi as the foremost technique to modulate gene expression (Unniyampurath et al., 2016). In the short-term, at least, it is likely that RNAi will continue to be used by researchers. This is particularly true for clinical applications, where more robust protocol for RNAi design and delivery make it easier to get trial approval. Additionally, some organisms – such as bacteria – lack the innate machinery required for RNAi, CRIRPRi can be used in a broader range of species.
Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Romero, D.A. and Horvath, P. (2007). CRISPR provides acquired resistance to viruses in prokaryotes. Science 315: 1709-1712.
Gilbert, L.A., Larson, M.H., Morsut, L., Liu, Z., Brar, G.A., Torres, S.E., Stern-Ginossar, N., Brandman, O., Whitehead, E.H., Doudna, J.A., Lim, W.A., Weissman, J.S. and Qi, L.S. (2014). CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154:442-451
Larson, M.H., Gilbert, L.A., Wang, X., Lim, W. A., Weissman, J.S. and Qi, L.S. (2013). CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nature Protocols 8:2180-2196
Shalem, O., Sanjana, N.E. and Zhang, F. (2015) High-throughput functional genomics using CRISPR-Cas9. Nature Reviews Genetics 16: 299-311
Tabernero, J., Shapiro, G.I., LoRusso, P.M., Cervantes, ., Schwartz, G.K., Weiss, G.J., Paz-Ares, L., Cho, D.C., Infante, J.R., Alsina, M., Goudner, M.M., Falzone, R., Harrop, J., Seila White, A.C., Toudjarska, I., Bumcrot, B., Meyers, R.E., Hinkle, G., Svrzikapa, N., Hutabarat, R.M., Clausen, V.A., Cehelsky, J., Nochur, S.V., Gamba-Vitalo, C., Vaishnaw, A.K., Sah, D.W., Gollob, J.A. and Burris, H.A. (2013) First-in-man trial of an RNA interference therapeutic targeting VEGF and KSP in cancer patients with liver involvement. Cancer Discovery 3:406-417
Unniyampurath, U., Pilankatta, R. and Krishnan, M.N. (2016). RNA interference in the age of CRISPR: Will CRISPR interfere with RNAi? International Journal for Molecular Sciences 17: