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Although SpCas9 is the most popular nuclease, Cas enzymes derived from other bacterial species have been described for editing applications. Commercially available alternatives to SpCas9 include the Acidaminococcus and Lachnospiraceae Cas12a (also known as Cpf1), from IDT and NEB respectively (Zetsche et al. 2015), the Staphylococcus aureus SaCas9, from Takara Bio (Ran et al. 2015), and the Francisella novicida FnCas9 from Merck (Acharya et al. 2019). The primary difference between Cas9 nucleases derived from different bacteria is in the protospacer adjacent motif (PAM) sequence that they require for binding and cleavage. For example, SaCas9 recognizes a longer PAM, 5'‐NNGRRT‐3', compared with 5'‐NGG‐3' for SpCas9. In addition, SaCas9 is about 1 kb smaller in size than SpCas9, so it can be packaged into viral vectors more easily, offering possibilities for non‐integrative AAV‐mediated gene therapy (De Caneva et al. 2019; Ginn et al. 2020).

Cas12a has recently emerged as an interesting alternative to SpCas9, due to its ability to target T‐rich motifs with the PAM, typically 5′‐TTTV‐3′, located upstream of the spacer. This makes Cas12a attractive as an epigenome editing platform, because it can target regions around transcription starting sites, which are inaccessible to SpCas9 (Tak et al. 2017). Although its potential in human research has yet to be fully realized, Cas12a has shown remarkable versatility in genome editing across a range of model organisms, including mice, porcine (female embryos), frogs (Xenopus), zebrafish, bacteria, and plants (Safari et al. 2019).