Cas9 is a bacterial RNA-guided endonuclease that uses base pairing to recognize and cleave target DNAs with complementarity to the guide RNA. In type II systems, Cas9 participates in the processing of spacer acquisition and crRNAs maturation, and is responsible for the destruction of the target DNA (Fig 1) and so on. The function of Cas9 in both of these steps relies on the presence of two nuclease domains: an HNH-like nuclease domain that cleaves the DNA strand complementary to the guide RNA sequence (target strand), and an RuvC-like nuclease domain responsible for cleaving the DNA strand opposite the complementary strand (non-target strand). To achieve site-specific DNA recognition and cleavage, Cas9 functions in conjunction with CRISPR RNAs (crRNAs) and a separate trans-activating crRNA (tracrRNA), which is partially complementary to the crRNA to mediate sequence-specific immunity against bacteriophages and other mobile genetic elements.
Upon infection, short phage sequences known as spacers, that integrate between CRISPR repeats and constitute a memory record of infection. Spacers are transcribed into small CRISPR RNAs (crRNAs) that identify the viral targets (protospacers) of the Cas9 nuclease. Cas9 was previously found to be the nuclease responsible for invader DNA cleavage in Type II systems. Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of an NGG protospacer adjacent motif (PAM) sequence immediately downstream of the target. In recent few years, scientists have found another important role of Cas9 protein: Be required for spacer acquisition. Cas9 specifies functional PAM sequences during spacer acquisition.
Fig 1. Cas9 protein function diagram.
Fig 2. Cas9 protein function in destruction of target DNA.
Cas9 associates with other Cas proteins involved in spacer acquisition. Cas9 forms a stable complex with Cas1, Cas2 and Csn2 that presumably participates in the selection of new spacers. The nuclease activity of Cas1, but not the RuvC- and HNH-based nuclease activities of Cas9, and also the Cas9 PAM-binding properties are required for new spacer acquisition. Cas9 specifies PAM sequences to ensure the acquisition of functional spacers during CRISPR adaptation. The key residues involved in Cas9 PAM recognition are not required for spacer acquisition, but they are necessary for the incorporation of new spacers with the correct PAM sequence. The non-specific DNA binding property of Cas9 is sufficient for spacer acquisition, but not for the selection of functional spacers.
Cas9 is shown above to be required during the phase of adaptation for the selection of spacers by recognizing the PAM of the protopacers. Cas9 and trans-activating crRNA (tracrRNA), which is a small trans-encoded RNA, are also play improtant roles in the maturation of crRNA , which is specifically found in the CRISPR repeat/spacer library (loci) of prokaryotes as one long string of crRNAs. The crRNA is made of the spacer (complementary to the target) and a structural piece (the repeat) that complements with the tracrRNA. The 20-nt spacer sequence of crRNA confers DNA target specificity, and the tracrRNA plays a crucial role in Cas9 recruitment.
In Type II systems, the precursor transcript of the CRISPR repeat-spacer array forms duplexes with the trans-activating tracrRNA through pre-crRNA repeat: tracrRNA anti-repeat interactions. The duplex RNAs stabilized by the protein Cas9 are recognized and cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid. The mature dual tracrRNA:crRNAs complexed with the protein Cas9 form the interference complex that target and cleave site specifically double-stranded DNA. The crRNAs can't bind to Cas9 alone, they have to complex with tracrRNAs, also known as the handle or scaffold region. Only then can the RNA-RNA duplex properly fit into Cas9.
Once Cas9 binds the sgRNA, the complex is ready to search for complementary target DNA (tDNA) sites. Target search and recognition require both complementary base pairing between the 20-nt spacer sequence and a protospacer in the tDNA, as well as the presence of conserved PAM sequence adjacent to the target site. Single mutations in the PAM can disable Cas9 cleavage activity. Cas9 initiates the tDNA search process by probing for a proper PAM sequence before interrogating the flanking DNA for potential guide RNA complementarity. Target recognition occurs through three-dimensional collisions, in which Cas9 rapidly dissociates from DNA that does not contain the proper PAM sequence, and dwell time depends on the complementarity between gRNA and adjacent DNA when a proper PAM is present. Once Cas9 has found a target site with the proper PAM, it triggers local DNA melting at the PAM-adjacent nucleation site, followed by RNA strand invasion to form an RNA–DNA hybrid and a displaced DNA strand from PAM-proximal to PAM-distal ends.
Upon PAM recognition and subsequent RNA–DNA duplex formation, Cas9 enzyme is activated for DNA cleavage. Cas9 protein contains two nuclease domains: a well-conserved RuvC domain consisting of three split RuvC motifs and an HNH domain that resides in the middle of the protein. Each domain cleaves one strand of the target dsDNA at a specific site 3bp from the NGG PAM sequence to produce a predominantly blunt-ended double strand break (DSB) (Fig 2). Cas9 nickases, however, cut only one strand of the DNA duplex, resulting in a single-strand break. When paired with sense and antisense sgRNAs targeting opposite strands, such Cas9 nickases can make staggered cuts within the tDNA and thus create a double nick-induced DSB for enhanced genome-editing specificity.
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