SpCas9-NLS is an RNA-guided endonuclease originating from Streptococcus pyogenes. This CRISPR-Cas9 system directs the enzyme to specific DNA targets through a guide RNA complex, which comprises crRNA and tracrRNA (or their chimeric single-guide RNA/sgRNA configuration). The nuclease recognizes genomic loci by complementary base pairing between the sgRNA spacer sequence and the target DNA, with PAM (protospacer adjacent motif) dependency located 3 nucleotides upstream of the cleavage site. Notably, the NGG triplet (5'-NGG-3') serves as the canonical PAM recognized by this system. For enhanced intracellular delivery and transcriptional efficiency, the recombinant protein is engineered with an N-terminal nuclear localization signal (NLS) derived from the simian virus 40 (SV40) T antigen. Beyond its primary application in genome editing, SpCas9-NLS is widely employed in vitro for targeted DNA cleavage and molecular cloning protocols.