degradation, with Cas1‐Cas2 identifying new protospacers for integration (Hille et al. 2018). It remains unclear how prespacers are extracted when found, but it is not inconceivable that the same machinery involved in processing prespacers is involved as well.
3.4 CRISPR Systems as the Basis for New Tools in Drug Discovery
So far, how different CRISPR systems exert their function and safeguard microbes from invading genetic elements have been discussed. In the last part of this Chapter, we will turn to how the understanding of CRISPR biology has advanced biotechnology.
3.4.1 Cas Proteins for Gene Editing
The most prominent application of CRISPR systems since its discovery has been genome editing. This has stemmed from realization that Cas proteins can induce precise cuts in DNA (Marraffini and Sontheimer 2008; Garneau et al. 2010) guided by the crRNA molecule (Gasiunas et al. 2012; Jinek et al. 2012). The first protein shown to do this in a simple manner was the type II Cas9 protein of S. pyogenes. The simplicity of type II systems, where only one effector protein guided by crRNA:tracrRNA is required to introduce a guided DNA break, in contrast to type I systems which require a dozen of Cas proteins to form a functional Cascade complex, has allowed this system to be used in a plethora of eukaryotic systems to introduce precise genetic changes (Cong et al. 2013; DiCarlo et al. 2013; Ding et al. 2013; Friedland et al. 2013; Hwang et al. 2013; Jinek et al. 2013; Mali et al. 2013). The system has been further simplified by fusing crRNA to tracrRNA into a chimeric single‐guide RNA (sgRNA) (Jinek et al. 2012), thus reducing the complexity of the toolkit needed for gene editing to just two components, where the sgRNA can very easily be replaced, allowing unprecedented flexibility in gene editing. SpyCas9‐mediated gene editing has since been one of the most prolific technologies in biomedical sciences, with the application ranging from precise (epi)genome engineering, modulation of gene activity, forward genetic screens, proteomics, imaging studies, diagnostics, and therapy.
While gene editing will be addressed in detail elsewhere in this book, we would like to give the basics here. SpyCas9, guided by crRNA:tracrRNA (or more frequently sgRNA), once heterologously expressed in a cell, tissue, or organism, is guided to a specific site in the genome where upon successful recognition it will introduce a blunt‐ended DSB. The repair outcome of the DSB by the host machinery can result in mutagenic events such as insertions or deletions by the nonhomologous end joining or microhomology‐mediated end joining (optimal for generating genetic knockouts), or stimulate the introduction of desired DNA sequence via recombination with a donor sequence (desired outcome for model generation and therapy). It is therefore of utmost importance for Cas9 to specifically and efficiently introduce a break only at the desired site. Furthermore, the precision and efficiency of other gene editing methods, such as base editing (Komor et al. 2016), prime editing (Anzalone et al. 2019), or site‐specific transposition (Chen and Wang 2019), are also in part dictated by the properties of Cas protein.
As discussed previously, Cas9 binds and identifies its target by recognizing the PAM sequence and then base pairing the crRNA initially with the seed sequence, and subsequently with the remainder of the sequence, followed by activation of the nucleolytic activities of RuvC and HNH domains. A plethora of biochemical studies have unequivocally confirmed that mismatches between seed and crRNA sequence are refractory to Cas9 editing (Jinek et al. 2012). However, mismatches outside the seed sequence (i.e. 10–20 nt away from the PAM) are well tolerated and support efficient cutting (Anderson et al. 2015), allowing Cas9 to cleave at off‐target sites. Potential off‐target mutagenesis induced by Cas proteins is a serious concern in therapy and model development, in particular, as Cas9 was shown to be able to induce large deletions and chromosomal rearrangements (Kosicki et al. 2018). In order to circumvent such problems, a number of different computational and experimental methods have been developed to identify and prevent off‐target modifications (refer to Chapter 20).
3.4.1.1 Cas9 Variants
A major strategy to reduce off‐target editing is to increase the specificity of Cas9 protein. To this end, SpyCas9 has been mutated at strategic residues so that mismatches between crRNA and the target strand are not compatible anymore with activating the enzyme’s nucleolytic activity, generating Cas9 variants (such as eSpCas9, HypaCas9, and SpCas9‐HF1) with improved specificity (Slaymaker et al. 2016; Kleinstiver et al. 2016a; Chen et al. 2017). However, improved specificity was often shown to have a negative impact on enzyme activity, decreasing the overall editing efficiency (Jones et al. 2020, Schmid‐Burgk et al. 2020), likely due to the altered interaction between sgRNA and Cas9 protein.
Another major limitation of using SpyCas9 for genome editing is the PAM sequence. As discussed previously, type II effector proteins require a compatible PAM sequence downstream of the protospacer sequence. The preferred PAM for SpyCas9 is 5’‐NGG‐3’, albeit it is able to use other GC‐rich PAMs at lower efficiencies (Leenay et al. 2016). To expand the targeting repertoire, Cas9 variants have also been evolved to be able to use other PAM sequences. Initial variants relied on structural understanding of the SpyCas9 mechanism, and have therefore mutated the key residue (Arg1335) involved in recognition of the third G in the PAM, followed by compensatory mutations in the vicinity that allowed these novel proteins SpyCas9‐VQR and ‐EQR to use the NGAN and NGNG as PAMs (Kleinstiver et al. 2015). A similar strategy was used to generate two other variants, the QQR1 which uses NAAG (Anders et al. 2016), and SpyCas9‐NG requiring a dinucleotide 5’‐NG‐3’ (Nishimasu et al. 2018). Recently, thanks to sophisticated in vitro evolution systems based on continuous phage‐assisted evolution, multiple SpyCas9 variants that recognize non‐G PAMs have been evolved; these variants can use NRN and to a lesser extent NYN PAM (Walton et al. 2020) and NRNH (where R represents A or G, Y is C or T, and H is A, C or T) (Miller et al. 2020). The development of these novel variants, albeit compromising their efficiency, brings us one step closer to a Cas system able to truly target any genomic sequence without restrictions imposed by the PAM.
3.4.1.2 Cas9 Orthologs
While in vitro evolution of SpyCas9 has expanded its utility, all the current variants still require a guanine in the PAM sequence or remain poorly active when using non‐G PAM, making the variants of this enzyme largely unsuitable for targeting AT‐rich sequences. Furthermore, while mutagenesis of Cas9 has improved its specificity, due to the structural constraints, these cannot be evolved past certain properties. Moreover, the size of the protein is of relevance for therapeutic applications, as typical vectors used for nuclease delivery (AAV) have a packaging capacity very close to the size of the SpyCas9 expression module (typically ~4.7 kb (Grieger and Samulski 2005)), making the use of this system difficult.
An alternative to in vitro evolution of Cas9 toward different activities, specificities, and sizes is to use Cas9 proteins originating from different species. Due to a staggering sequence variation, Cas9 from different species exhibit diverse specificities, activities, thermodynamic properties (i.e. exhibit longer stability and activity at higher temperatures), and PAM requirements. For example, in parallel with the discovery of SpyCas9 and the interference mechanisms, two orthogonal Cas9 from Streptococcus thermophilus (StCas9) has been described (Magadan et al. 2012; Karvelis et al. 2013), that were later shown to display editing efficiencies comparable to SpyCas9 in human cells, but with substantially lower off‐target rate and longer, more diverse PAMs (NNAGAAW and NGGNG, where W is A or T) (Cong et al. 2013; Muller et al. 2016). Over the course of the last decade, a number of different, often smaller Cas9 proteins have been described and then used in genetic engineering in eukaryotic cells. These include Cas9 proteins from S. aureus (SauCas9) with the NNGRRT PAM (Ran et al. 2015), Neisseria meningitidis Nme1Cas9 with NNNNGATT PAM and lower off‐target due to longer crRNAs (Esvelt et al. 2013; Hou et al. 2013; Zhang et al. 2013), and also Nme2Cas9 requiring NNNNCC (Edraki et al. 2019), Staphylococcus auricularis (SauriCas9) requiring NNGG in PAM (Hu et
