Cas12b proteins to specifically and efficiently edit eukaryotic genomes (Strecker et al. 2019a; Teng et al. 2019a; Teng et al. 2019c; Ming et al. 2020). A Cas12b worth mentioning originates from Alicyclobacillus acidiphilus (AaCas12b), which has unprecedented specificity where the enzyme is not able to tolerate any mismatches (Teng et al. 2018).
A recent discovery of Cas12e from Deltaproteobacteria (formerly known as DpbCasX) (Burstein et al. 2017) has already led to a demonstration of exquisite editing activity in human cells (Liu et al. 2019a). Very recently, a novel collection of compact type V Cas effector proteins have been identified in genomes of Biggiephage clade of huge phages. Cas12j (also known as CasΦ) was shown to possess some superb features: the size of the effector protein is very small compared with commonly used Cas9 proteins (only ~70 kDa compared to 160 kDa), a T‐rich PAM (TBN, where B is G, T, or C), is able to process crRNA with its RuvC domain, generating staggered‐end break end with 8–12 nt‐long 5’ overhang, and with potent activity in human cells (Pausch et al. 2020). With emphasis placed on the discovery of miniature class 2 effectors, a group of Cas12f proteins was recently biochemically characterized and orthologs with interesting properties, suggesting that there are many more CRISPR‐Cas systems to be harnessed for gene editing (Karvelis et al. 2020).
Finally, a number of different CRISPR systems have been developed to perform Transposon‐assisted site‐specific integration. As discussed previously, many cas genes are frequently found with transposons in the same operon, with concurrent loss of essential interference machinery, such as many type IV and V systems. A spectacular example is Cas12k, which was able to support transposition of a 10 kb insert at crRNA‐directed site when heterologously expressed in E. coli with the missing components (Strecker et al. 2019b). Similarly, the Tn6677 transposase of Vibrio cholerae has co‐opted the type I‐F machinery for transpositions; this has very recently allowed development of highly efficient CRISPR‐guided transposition in bacteria (Klompe et al. 2019; Vo et al. 2021), with the potential to be of major use in vertebrate genome editing.
3.4.2 Application of Cas Proteins Beyond Genome Editing
3.4.2.1 dCas9 Fusions
While Cas9 proteins were originally used for gene editing purposes, realization that mutating two key residues in RuvC and HNH domain (D10A and H840A, respectively) that abrogate its catalytic activity but not its binding (Jinek et al. 2012; Qi et al. 2013) generates a sequence‐specific sgRNA‐directed DNA binding protein has opened a new range of application of the bacterial immune system. By fusing a specific protein domain to catalytically deficient Cas9 (dCas9) allows one to direct a protein activity to specific genomic loci. This approach has been brought to life by fusing dCas9 to repressive transcriptional domains such as KRAB domain of Kox1 or the chromoshadow domain of HP1α (Figure 3.7a), producing a robust 25–100‐fold reduction of targeted gene expression without any direct off‐targets (Gilbert et al. 2013; Gilbert et al. 2014), generating the strategy called CRISPR interference (CRISPRi). Similarly, a complementary approach named CRISPR activation (CRISPRa) was developed by fusing transactivator domains of VP64 or NFkB (Perez‐Pinera et al. 2013; Gilbert et al. 2014) (Figure 3.7a). Both of these systems have been improved further, namely by identifying protein domains with optimal activities (Chavez et al. 2016) or improving the output by recruiting multiple synergistic effectors (Tanenbaum et al. 2014; Zalatan et al. 2015). In parallel, dCas9 system for introducing specific epigenetic modifications at programmed genomic loci was also developed by fusing dCas9 to catalytic domains of DNA methyltransferases and various histone‐modifying enzymes (Figure 3.7c, d), allowing one to enforce a heritable modulation of gene expression (Amabile et al. 2016; Liu et al. 2016; Vojta et al. 2016). While these systems are not as nearly as sophisticated as CRISPRi and CRISPRa, they present a promising basis to therapeutic epigenome editing. dCas9 systems have also been used for a number of other purposes used to elucidate biology, such as imaging and proteomics Figure 3.7e; we refer the reader to excellent reviews on these recently developed systems (Xu and Qi 2019). It should be also noted that dCas9 can be used to tether DNA‐modifying enzymes (such as cytidine or adenosine deaminases) which can alter the underlying sequence; this is the basis for DNA base editing, which is addressed in detail in Chapter 14.
Figure 3.7 Applications of CRISPR systems beyond genome editing. Fusing catalytically inactive Cas9 (dCas9) to a transcriptional activator (a) or repression (b) domains can directly regulate gene expression. By fusing dCas9 to DNA‐modifying enzymes, such as DNA methyltransferases (DNMT) or TET enzymes (c), or histone deacetylases (HDACs), histone acetyltransferases (HATs), or histone methyltransferases (HMTs) (d), one can modify the epigenetic marks leading to indirect albeit potentially permanent modulation of gene expression. If fused to a fluorescent protein, dCas9 can be used to visualize targeted locus in imaging studies (e). RNA targeting enzymes such as Cas13a (f) are able to specifically degrade target RNA molecules, or by fusing a catalytically inactive version of this enzyme to RNA‐modifying enzymes (such as ADAR deaminases), one can perform RNA editing; here, deamination of adenosine to inosine (decoded as a guanosine) is depicted (g). Cas13‐based detection methods rely on the activation of Cas13a by recognizing the target RNA molecule, which is then unspecifically able to digest the reporter RNA molecule. Cleavage of the reporter molecule separates fluorophore (F) from the quencher dye (Q), generating a diagnostic fluorescent signal (h). A similar approach can be used for DNA‐targeting enzymes exhibiting collateral activity, such as some Cas12 enzymes.
3.4.2.2 RNA Targeting
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