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Technologies based on CRISPR-Cas systems can be used not only for genome editing but also for controlling gene expression. They can target genomic regulatory elements to specific DNA regions, temporarily turning the genes on and off, which allows the scientists to study the gene's function in more detail, especially when it comes to essential genes.
CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) methods take advantage of nuclease-deactivated dCas9 (usually with D10A and H840A point mutations), where "d" stands for "dead". While dCas9 cannot make DSBs, it can still target a specific DNA sequence when loaded with an appropriate gRNA. dCas9 can be fused/interact with one or several transcription effectors (transcription activators or inhibitors), and thus control transcription. dCas9 commonly used for this purpose is S. pyogenes dCas9.
In the case of CRISPRa, dCas9 is fused to transcription activator(s), most commonly VP64 (Fig.11), which consists of four herpes simplex virus VP16 molecules. p65 (NF-κB transcription factor) and RTA (EBV immediate-early transcription activator) are also often attached to the dCas9-Vp64 complex in order to activate the transcription of the target gene. The CRISPRa system employing those 3 molecules is called dCas9-VPR. Numerous other transcription activator combinations can also be used in CRISPRa. While dCas9 fused to transcription effector(s) can be directed to the promoter region by a regular sgRNA, special gRNAs that can recruit (attach to) extra activation domains can also be used.
In the case of CRISPRi, the gRNA loaded inside dCas9 complexes with the Krüppel-associated box (KRAB) domain. dCas9-KRAB induces heterochromatin formation at the target promoter, effectively reducing transcription of the target gene. In bacteria, dCas9 alone can also be used to reduce transcription. However, in the case of eukaryotic cells, fusion with auxiliary transcription inhibitors is often required to achieve desired levels of repression.
Figure 1. Various transcription effectors used CRISPRa and CRISPRi
Transcriptional activation and repression are both reversible processes. There are multiple variants of regulatory domains that can be fused to dCas9, including epigenetic DNA modifiers. dCas can also be fused to fluorescent proteins such as GFP for in vivo imaging of certain genomic regions, which aids the research of chromosomal organization and dynamics.
Cas12a can also be redesigned to dCas12a (e.g., Acidaminococcus sp. dCas12a) by deactivating its RuvC domain (E993A mutation) and used similarly to dCas9. In the case of multi-gene repression, dCas12a requires the expression of a single CRISPR array only, while dCas9 requires independent expression of several sgRNAs. dCas12a still exhibits RNase activity, thanks to which it can process a precursor CRISPR array and create multiple mature crRNAs in vivo (in E. coli). This ability could make dCas12a CRISPRi a more convenient tool than dCas9 in multiplex gene regulation.
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Figure 1: Gebre, Makda & Nomburg, Jason & Gewurz, Benjamin. (2018). CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions. Viruses. 10. 55. 10.3390/v10020055
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