Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 48

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.