Genome Editing in Drug Discovery. Группа авторов

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СКАЧАТЬ engineering further relieving the PAM constraints to YG) (Hirano et al. 2016), Campylobacter jejuni (CjCas9) with NNNNRYAC (Kim et al. 2017), Streptococcus canis (ScCas9) with NNG (Chatterjee et al. 2018), Geobacillus stearothermophillus (GeoCas9) using the NNNNCRAA as a PAM (Harrington et al. 2017), and Streptococcus macacae (SmacCas9) requiring a NAAN in PAM site (Chatterjee et al. 2020).

      Recently, a biochemical tour de force has identified and characterized the activity of 79 novel Cas9 proteins. Previously unknown G‐, A‐, T‐, C‐rich PAM repertoires, together with different patterns of cuts (blunt or with staggered ends), kinetics, and crRNA sequences have been described, forming the basis of a catalogue of orthologs that can be used for genome editing in the future (Gasiunas et al. 2020). Together with the ever‐expanding and ever‐improving number of variants with a wide range of PAM specificities (Collias and Beisel 2021), one can envisage that thanks to these efforts one will be able to choose a Cas9 protein for genome editing purposes based on the desired target sequence, unconstrained by the PAM restrictions, specificities, and activities.

      3.4.1.3 The Use of Other Cas Proteins in Genome Editing

      In parallel with the advent of new SpyCas9 variants and Cas9 orthologs, there was a push to examine whether other types of CRISPR‐Cas9 proteins can be used for gene editing. While type I systems were essentially the first CRISPR systems to be examined in detail (Barrangou et al. 2007), the fact that the system requires a multitude of proteins (at various stoichiometry) gave little appeal to use this technology in eukaryotic systems. Type I systems are extensively used for precise engineering in microbes (Kiro et al. 2014; Li et al. 2016; Pyne et al. 2016; Xu et al. 2019), and only recently have been implemented for editing in human cells. Due to type I system’s reliance on hyperactive Cas3 helicase/nuclease, editing by the Cascade complex leads to large deletions (up to 100 kb) from a single cut site (Dolan et al. 2019; Morisaka et al. 2019; Osakabe et al. 2020). Whereas using type I systems are therefore an excellent tool for deleting large segments of the genome (which could be useful for removing transgenes from model organisms or interrogating regulatory elements, for example), this system has been adapted for introducing small insertions by fusing the Cascade complex to FokI endonuclease domain. When Cas3 was omitted from and FokI‐Cascade fusion expressed to target proximal sequences, DNA sequences were successfully deleted (Cameron et al. 2019). Whereas the efficiency of Cascade‐FokI mediated editing is low compared with standard SpyCas9, it is on par with other genome editing approaches using catalytically inactive Cas proteins (see Section 3.4.2).

The most prominent use of other types of CRISPR systems is the Cas12a (originally named Cpf1), a member of type V systems (Zetsche et al. 2015), which have some beneficial features over Cas9 proteins. The first three described Cas12a proteins, the Franscisella novicida (FnCas12a), Acidaminococcus sp. (AsCas12a), and Lachnospiraceae bacterium (LbCas12a), use T‐rich PAM sequences, such as TTN and TTTN, making them a complementary tool to G‐rich PAM utilizing Cas9 proteins. Secondly, Cas12a proteins are able to autonomously process crRNA from pre‐crRNA, unlike Cas9 proteins which require tracrRNA (Deltcheva et al. 2011). This property has been exploited to significantly improve simultaneous genome editing at multiple sites, where multiple Cas12a‐compatible crRNAs are expressed as a single pre‐crRNA transcript, contrasting to Cas9 multiplex editing which requires multiple expression modules, one for each sgRNA (Zetsche et al. 2017; DeWeirdt et al. 2021). The most prominent benefit of Cas12a is, however, its cleavage mechanism, where Cas12a generates staggered ends (unlike blunt ends generated by most of Cas9 proteins) outside the critical seed region. Staggered ends are particularly suitable for precise integration of DNA via NHEJ or MMEJ (Maresca et al. 2013). As Cas12a cuts the target DNA away from the seed sequence, repair via NHEJ (leading to small indels) will still support cleavage. Cas12 editing is therefore likely to promote resection of the staggered DNA break, leading to deletions spreading into the seed region or more favorably promoting HDR and MMEJ (Begemann et al. 2017; Moreno‐Mateos et al. 2017; Li et al. 2018a). Finally, Cas12a shows lower tolerance to mismatches, with only the mismatches at the last 4 nt of a 23 nt‐long crRNA supporting cleavage (Kleinstiver et al. 2016b), in contrast to SpyCas9 which will cut DNA even with 10 mismatches (Klein et al. 2018; Jones et al. 2020). Similarly to Cas9 systems, new Cas12a orthologs have also been described to expand the targeting spectrum by choosing Cas12a variants with convenient PAM requirements, such as those of Coprococcus eutactus (CeCas12a) or F. novicida (FnCas12a) (Aliaga Goltsman et al. 2020; Chen et al. 2020; Zetsche et al. 2020). Furthermore, AsCas12a has been evolved for higher specificity and activity (Kleinstiver et al. 2019).

      Other type V systems have very recently been adapted for gene editing. Type V‐B systems, epitomized by Cas12b effector proteins, were difficult to use for gene editing due to their collateral ssDNAse activity and low activity at mammalian physiological temperature. Recent engineering and repurposing efforts have allowed a set of evolved 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 СКАЧАТЬ