The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system is currently the most commonly used RNA-Guided Endonuclease technology for genome engineering. There are two distinct components to this system: (1) a guide RNA and (2) an endonuclease, in this case the CRISPR associated (Cas) nuclease, Cas9. The gRNA/Cas9 complex is recruited to the target sequence by the base-pairing between the gRNA sequence and the complement to the target sequence in the genomic DNA. For successful binding of Cas9, the genomic target sequence must also contain the correct Protospacer Adjacent Motiff (PAM) sequence immediately following the target sequence.
Function:
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). 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) (Probable). 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. Subsequently Cas9/crRNA/tracrRNA endonucleolytically cleaves linear or circular dsDNA target complementary to the spacer. The target strand not complementary to crRNA is first cut endonucleolytically, then trimmed by 3'-5' exonucleolytically. DNA-binding requires protein and both RNA species. Cas9 probably recognizes a short motif in the CRISPR repeat sequences (the PAM or protospacer adjacent motif) to help distinguish self versus nonself.
Subunit:
Monomer. Binds crRNA and tracrRNA.
Similarity:
Belongs to the CRISPR-associated protein Cas9 family.
Subtype II-A subfamily.
SWISS:
Q99ZW2
Gene ID:
901176
Database links:
Entrez Gene: 901176 Streptococcus pyogenes
SwissProt: Q99ZW2 Streptococcus pyogenes
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Sample:
Lane 1: CRISPR-Cas9 transfected 293 cells lysate
Lane 2: Non-transfected 293 cells lysate
Primary: Anti-CRISPR-Cas9 (SLM52042M) at 1/5000 dilution
Secondary: Goat Anti-Mouse IgG - HRP at 1/5000 dilution
Predicted band size: 160 kD
Observed band size: 150 kD
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