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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 al. 2020), Francisella novicida (FnCas9) with NGG PAM (with rational 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.