CRISPR-Cas9

Has 2 endonuclease domains. The discontinuous RuvC-like domain (approximately residues 1-62, 718-765 and 925-1102) recognizes and cleaves the target DNA noncomplementary to crRNA while the HNH nuclease domain (residues 810-872) cleaves the target DNA complementary to crRNA (PubMed:22745249, PubMed:24529477).

Has a bilobed architecture with a recognition lobe (REC, residues 60-718) and a discontinuous nuclease lobe (NUC, residues 1-59 and 719-1368) (PubMed:24505130, PubMed:24529477). The crRNA-target DNA lies in a channel between the 2 lobes (PubMed:24529477, PubMed:26841432). Binding of sgRNA induces large conformational changes further enhanced by target DNA binding (PubMed:26113724, PubMed:26841432). REC recognizes and binds differing regions of an artificial sgRNA in a sequence-independent manner. Deletions of parts of this lobe abolish nuclease activity (PubMed:24529477).

The PAM-interacting domain (PI domain, approximately residues 1099-1368) recognizes the PAM motif; swapping the PI domain of this enzyme with that from S.thermophilus St3Cas9 (AC Q03JI6) prevents cleavage of DNA with the endogenous PAM site (5'-NGG-3') but confers the ability to cleave DNA with the PAM site specific for St3 CRISPRs.

CRISPR (clustered regularly interspaced short palindromic repeat) is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements and conjugative plasmids) (PubMed:21455174). CRISPR clusters contain spacers, sequences complementary to antecedent mobile elements, and target invading nucleic acids. CRISPR clusters are transcribed and processed into CRISPR RNA (crRNA). In type II CRISPR systems correct processing of pre-crRNA requires a trans-encoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc) and this protein. The tracrRNA serves as a guide for ribonuclease 3-aided processing of pre-crRNA; Cas9 only stabilizes the pre-crRNA:tracrRNA interaction and has no catalytic function in RNA processing (PubMed:24270795). Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer; Cas9 is inactive in the absence of the 2 guide RNAs (gRNA). The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed 3'-5' exonucleolytically. DNA-binding requires protein and both gRNAs, as does nuclease activity. Cas9 recognizes the protospacer adjacent motif (PAM) in the CRISPR repeat sequences to help distinguish self versus nonself, as targets within the bacterial CRISPR locus do not have PAMs. DNA strand separation and heteroduplex formation starts at PAM sites; PAM recognition is required for catalytic activity (PubMed:24476820). Confers immunity against a plasmid with homology to the appropriate CRISPR spacer sequences (CRISPR interference) (PubMed:21455174).

Belongs to the CRISPR-associated protein Cas9 family. Subtype II-A subfamily.

csn1, SPy_1046, cas9, CRISPR-associated endonuclease Cas9/Csn1, SpCas9, SpyCas9

• swissprot:Q99ZW2

Proteins

Immuno-oncology

158441Da