Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 45

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.