Abstract
The discovery and application of RNA-guided, Streptococcus pyogenes Cas9 (SpCas9) nucleases has revolutionized gene-editing platforms. However, the use of SpCas9 in recent research has revealed certain drawbacks, such as its high level of mutagenic activity, stringent protospacer adjacent motif (PAM) requirement, and large size. As more information becomes available for CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems, Cas9 has been engineered to modify its activity and target recognition residues with higher efficiency. For example, variants demonstrating enhanced target specificity, novel PAM recognition, genome- and epigenome-modifying activity, and base-editing capabilities have been designed in recent years. As the Cas toolbox expands, advantageous properties of Cas9 orthologs, such as smaller size or increased efficiency, are demonstrating equal or greater success in gene-editing applications compared to SpCas9. These variants are also being redesigned as enhanced or altered enzymes for more precise and efficient outcomes. This chapter seeks to review current knowledge of SpCas9, its variants, and Cas orthologs to optimize gene-editing protocols and therapeutic strategies.
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