SpCas9 is a large enzyme called a nuclease that is composed of over 1,000 amino acids, specifically 1,368. The process of SpCas9 going through discrete DNA interrogation steps before cleavage is managed by DNA interactions often with protein structal manipulation. This makes SpCas9 a sequence-specific DNA-binding protein. Sequence-specific DNA-binding proteins regulate gene expression by recognizing and binding defined nucleotide motifs within the genome. They strictly bind to particular DNA sequences depending on the base pair pattern. DNA-binding proteins can act either as a trasciptional enhancer or as a repressor, and in the case of SpCas9 it is a repressor. The SpCas9-gRNA complex locates its targets by simultaneously monitoring both DNA and SpCas9–gRNA at a high spatiotemporal resolution. SpCas9–gRNA begins the PAM search through running into DNA, and it speeds up the search, by reducing the time at non-PAM sites, interrogating only the PAM-line DNA for gRNA complements. SpCas9 specificity has been primarily characterized by using mismatched gRNA libraries, in vitro selection, and reporter assays. Cas9 is sensitive to the number, position, and distribution of the mismatches throughout the guide sequence, and it's important to consider both the NGG and NAG PAMs in off-target analysis because they both play a big role in off-target cleavage, thus playing a major role in Cas9's specificity.
SpCas9 is a multidomain DNA endonuclease containing two distinct lobes, one being the alpha-helical recognition (REC) lobe and the othe other being the nuclease lobe (NUS). Also present more variable C-terminal domain (CTD). The NUC lobe contains the HNH and RuvC nuclease domains that serve to cleave the Target and the Non-Target strands respectively. The SpCas9 structure is important because it provides a useful scaffold for engineering Cas9 and sgRNA, given that it is possible to introduce orthologous domain recombination, truncation, and peptide linkage.