Cas3

Thermophile Thermobifida fusca Cas3 is a four domain protein with an N-terminal HD-type nuclease domain, followed by two RecA-like domains forming a superfamily 2 helicase motif, then the Cas3 specific linker and C-terminal domain. Single stranded DNA is passed 3′-to-5′ from the helicase domain to the nuclease domain where it is hydrolysed. Nuclease activity Cas3 consists of an HD-nuclease domain fused to two RecA-like domains. These domains deliver ATP-dependent ssDNA translocation and degradation. An exposed helix and tyrosine/tryptophane residues located at the RecA-HD domain are essential for the functionality of this mechanism. Additionally, the nuclease function is supported by metal ions such as iron, manganese, and calcium, which work to regulate interactions between Cas3 and Cascade. The validation of target DNA sequences by Cascade is essential for the effectiveness of Cas3 as a nuclease in CRISPR systems. This process is achieved through the identification of a Protospacer Adjacent Motif at a target sight, followed by the formation of a Cascade R-loop that locks onto the target DNA. This structure allows Cas3 to load onto the ssDNA and allow for interactions with the Cse1 subunit of Cascade channels. Specifically, Cas3 docks to the alpha helix H1 of Cse1, triggering the degradation of targeted DNA by the Cas3 HD-nuclease activity. This degradation is achieved through DNA nicking by the HD-nuclease in the R-loop, which displaces ssDNA. RecA domains power the ATP-dependent translocation of Cas3 along ssDNA. == Functional implications ==

Gene editing Cas3-catalyzed DNA destruction plays an important role in genetic editing reactions. Cas3, when complexed with Cascade, can function in targeted genetic insertions and deletions when delivered into cells as proteins with nuclear localization signals. Due to the requirement for Cascade to lock on to the DNA target before Cas3 activation, there is a low rate of off-target effects. Cas3 has been shown to facilitate large regions of genetic deletion, up to 200kb, when expressed in human HEK293 cells. Additionally, the exon-skipping capabilities of Cas3 show promise for future genetic therapy treatments for conditions such as muscular dystrophy. CRISPR-mediated adaptive immunity CRISPR-mediated adaptive immunity requires three stages: sequence acquisition, CRISPR RNA biogenesis, and target interference. CRISPR RNA-guided surveillance complexes aid this process in efficiently targeting non-self through the recognition of short motifs. Type IE systems, which use Cas3, use a Cascade as the surveillance complex in this process. Through the binding and bending of Cascade to target DNA, R-loops are formed, and Cas3 is recruited to the complex. ATP enhances Cas3 recruitment to R-loops, and binding enhances Cas3 ATPase activity. The HD-nuclease domain of Cas3 nicks and unidirectionally degrades the target DNA of the displaced strand in the 3' to 5' direction. Cas3-mediated degradation may also recruit Cas1 and Cas2, proteins involved in the process of integrating spacers, priming for future foreign immunity. == References ==