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So far, we have discussed how various CRISPR systems can be used for genome editing. The discovery of type VI systems and their ability to target RNA by Cas13 effectors has led to the rise of novel approaches to manipulate the transcriptome of a given cell without altering the underlying genetic component. As discussed in previous sections, Cas13 proteins can degrade target RNA molecules by nucleolytic activity of their HEPN domain. Heterologous expression of Cas13 orthologs, such as those of Leptotrichia wadei (LwaCas13a), Leptotrichia shahii (LshCas13a) (Abudayyeh et al. 2017), Prevotella sp. P5‐125 (PspCas13b) (Cox et al. 2017) or Ruminococcus flavefaciens (RfxCas13d, also known as CasRx) (Konermann et al. 2018), and cognate crRNA in human cells leads to knockdown of specific RNA transcripts (Figure 3.7f) without substantial off‐target effects usually associated with short‐hairpin RNA (shRNA). The knockdown efficiency is ortholog and transcript dependent, but comparable to reduction observed with genome editing approaches (50–90%). As discussed previously, nearly all Cas13 proteins exhibit an indiscriminate RNase activity, meaning that they can degrade any bystander RNA. While this is an important mechanism of conferring population‐level immunity in prokaryotes (Meeske et al. 2019), the collateral activity of tested proteins has not been shown when expressed in human cells, encouraging further use of this system in mammalian models. It should be noted that some Cas9 proteins are also able to target RNA (Sampson et al. 2013; Dugar et al. 2018; Strutt et al. 2018), but their activities have not been tested yet in human cells.

Mutating the key catalytic residues of Cas13 converted this protein to a binding‐proficient but nuclease‐deficient protein (dCas13). Subsequent targeting to key regulatory pre‐mRNA elements (such as splicing acceptor or donor sites) permits one to alter the splicing pattern of target transcript (Konermann et al. 2018). Importantly, dCas13 can be used as a programmable RNA‐binding protein (analogous to dCas9), and fusing it to a suitable protein domain allows one to modulate or analyze the properties of target RNA. For example, fusing a domain of Adenosine deaminases acting on RNA (ADAR), key enzymes involved in RNA editing, allows one to post‐transcriptionally change the sequence of RNA from adenine to inosine (decoded during the translation as guanine), altering the protein primary sequence without affecting the genome (Cox et al. 2017) (Figure 3.7g).

Overall, the adaptation of RNA‐targeting type VI CRISPR systems now allows researchers to manipulate the transcriptome, a powerful and complementary tool to manipulating the genome by other systems.