1.4.2. Novel Variants of Cas Protein and Applications

In commonly used CRISPR-Cas9 systems, the *Streptococcus pyogenes* (SpCas9)-gRNA complex generally recognises the region 20 nt upstream of the PAM sequence (5 -NGG-3 ). To broaden the Cas9 protein recognition sequence and reduce the off-target editing rate, several approaches have been used to broaden PAM compatibility and enhance specificity. These approaches are based on the structural characteristics of SpCas9 binding to gRNA and target DNA. For example, the Cas9 variants SpCas9-VQR (NGA-PAM), SpCas9-EQR (NGAG-PAM), and SpCas9-VRER (NGCG-PAM) functioned, but their cleavage activity levels were lower compared to that of the wild-type SpCas9 in *Arabidopsis* and rice [99–102]. SpCas9-NG has a broader recognition sequence with enhanced compatibility, recognising NG-PAM, and has successfully generated targeted mutations in rice and *Arabidopsis* [103–105].

The variants SpCas9-HF1, eSpCas9, and HypaCas9 have been developed to enhance Cas9 protein-cleavage specificity. They show reduced off-target editing activities, indicating high specificity in plant cells [106–108]. Cas9 protein-directed evolution has been developed for Cas9 engineering, conferring high-specificity engineered SpCas9 proteins such as xCas9 [109], evoCas9 [110], and Sniper-Cas9 [111]. xCas9 recognises the NG, GAA, and GAT PAM sequences. Although the gRNA containing these PAM sequences can be mutated in plants, the mutation efficiencies and the preferences for different corresponding PAM sequences differ between cells. For example, the cleavage activity of xCas9 is lower in rice callus than in mammalian cells [112], and xCas9 does not recognise the NG-PAM sequence in tomatoes [32]. Cas9-NG has a stronger cleavage activity than xCas9, especially at CGG, AGC, TGA, and CGT sequence recognition sites [98], making Cas9-NG more suitable for genome editing at the NG-PAM site in plants. The single-base editing system developed based on Cas9 variants (SpCas9-NG and SpCas9-VQR) has been applied to precise base-editing of plant genomes [103,113].

At present, the most commonly used Cas9 protein comes from *Streptococcus pyogenes*, in order to broaden the Cas9 protein recognition sequence, orthologous Cas9 proteins have been isolated from other bacteria; for instance, NmCas9 has been isolated from *Neisseria meningitidis* [114], SaCas9 from *Staphylococcus aureus* [115], StCas9 from *Streptococcus thermophilus* [116], FnCas9 from *Francisella novicida* [117], and CjCas9 from *Campylobacter jejuni* [118]. These proteins are smaller than SpCas9, which is an advantage in cassette delivery. In *Arabidopsis*, the SaCas9 and SpCas9 systems do not interfere with each other [119], so they can fully utilise Cas9 orthologues that recognise different PAM sequences. Such simultaneous targeting by Cas9 orthologues with different PAM sequences would enable multiplex genome engineering by simultaneously targeting more than one site.

With the continued discovery and investigation of the functions of CRISPR protein family members, new types of Cas proteins have been discovered including the type VI CRISPR-Cas system Cas13 (C2c2) protein, which recognises RNA sequences and exhibits RNA editing activity without altering the genome sequence [120,121]. This system has been successfully applied to knockout gene function in rice and tobacco and promote resistance to RNA viruses in *Arabidopsis* [121,122]. Likewise, the CRISPR-Cas13 system created RNA-guided immunity against RNA viruses in plants. Type V CRISPR-Cas systems, such as Cas12c, Cas12g, Cas12h, Cas12i, and Cas14, are distinguished according to the type of their target template (ssRNA, ssDNA, dsDNA, or ssDNA) and cleavage activity strength. Their functions range from dsDNA nicking and cleavage, and can have collateral cleavage activity on ssRNA, ssDNA, dsDNA, and ssDNA [123,124]. In short, the functional differentiation of these Cas protein variants can be used to target mutations in different nucleic acid types, induce a small number of 100 kb sequence deletions, and expand the repertoire of plant genome-editing tools.
