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CRISPR-associated protein (Cas) 12i Cas12i1 and Cas12i2 are class 2 type V-I (class 2, type V family, subtype I; Cas12i) CRISPR effector proteins. Class 2, type V family members, have names each which begin with Cas12, and predominantly cleave the non-target strand of a double-stranded DNA (dsDNA) substrate. Two Cas12i proteins (Cas12i1 and Cas12i2), have CRISPR RNA (crRNA)-guided dsDNA cleavage activity. Cas12i1 and Cas12i2 cleave dsDNA adjacent to protospacer adjacent motif (PAM) sequences, preferring a 5' TTN PAM. They cleave the non-target DNA (NTD) strand prior to the target DNA (TD) strand to generate staggered double-strand breaks; for Cas12i1 the NTD strand was cleaved 13-15 nucleotides after the PAM duplex, and the TD strand was cleaved 24 nucleotides after the PAM duplex; for Cas12i2 the NTD strand was primarily cleaved 31 nucleotides after the PAM duplex and the TD strand was cleaved 24 nucleotides after the PAM duplex. In addition to dsDNA cleavage, Cas12i1 and Cas2i2 possess ssDNA cleavage activity, and pre-crRNA processing activity for crRNA maturation. The class 2 effector modules are single large proteins, distinguished by their domain architectures into type II, V and VI systems. Type V is the most diversified type and is further divided into subtypes A-I, K, and CRISPR-CasPhi. These subtypes, with the exception of subtype K, have ssDNA cleavage activity; after the effector protein cleaves the targeted dsDNA substance, the PAM-distal product is released, but the Cas protein is still bound to the PAM-proximal strand; at this point, the RuvC domain can still access ssDNA substrates and perform non-specific cleavage (also known as the promiscuous cleavage of collateral ssDNA or trans-cleavage. The type II effectors (Cas9) contain two nuclease domains that are each responsible for the cleavage of one strand of the target DNA, with the HNH nuclease inserted inside the RuvC-like nuclease domain sequence. By contrast, the type V effectors (Cas12) only contain a RuvC-like domain that cleaves both strands. Both type II and type V effectors adopt a similar architecture comprising a recognition lobe (REC) and a nuclease lobe (NUC). Cas9 endonucleases employ both the RuvC and HNH domains for DNA cleavage, however, Cas12 endonucleases use a single RuvC domain for DNA cleavage. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated protein) is an RNA guided adaptive immunity system present in bacteria and archaea, which defends its host against foreign nucleic acids that originate from phages or plasmids. The CRISPR-Cas-mediated adaptive immune response can be divided into three steps, including the acquisition of spacer derived from invading nucleic acids, crRNA processing, and target degradation. CRISPR-Cas systems are divided into two classes (1 and 2) and six types (class 1: types I, III and IV; class 2: types II, V and VI). |
Jiyao W et al.(2023). "The conserved domain database in 2023.",