CRISPR type V

SEE CRISPR-Cas The prototype type V effector Cpf1 (subtype V-A) contains only one nuclease domain (RuvC-like) that is identifiable by sequence analysis. However, analysis of the recently solved structure of (from Acidaminococcus sp. BV3L6, 5b43) has revealed a second nuclease domain, the fold of which is unrelated to HNH or any other known nucleases. In analogy to the HNH domain in Cas9, the , and it is responsible for cleavage of the target strand. Screening of microbial genomes and metagenomes for undiscovered class 2 systems has resulted in the identification of three novel CRISPR-Cas variants. These include subtypes V-B and V-C, which resemble Cpf1 in that their predicted effector proteins contain a single, RuvC-like nuclease domain. Cleavage of target DNA by the type V-B effector, denoted C2c1, has been experimentally demonstrated. Subtype V-A (Cpf1) Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA The overall structure of the (from Acidaminococcus sp. BV3L6, 5b43). The structure revealed that consisting of an α-helical and a , with the bound to the . . The , whereas the . The , and the . play functional roles similar to those of the WED (Wedge) and PI (PAM-interacting) domains of Cas9 (see CRISPR-Cas9), respectively, although the two domains of AsCpf1 are structurally unrelated to the WED and PI domains of Cas9. is involved in DNA cleavage (described below). Thus, domains A, B, and C are referred to as the WED, PI, and Nuc domains of Cas9, respectively. The in the Cpf1 sequence. The comprises seven α-helices and a β-hairpin. The that form the endonuclease active center. A characteristic helix (referred to as the bridge helix, ) is located between the RuvC-I and RuvC-II motifs and connects the REC and NUC lobes. The is inserted between the RuvC-II and RuvC-III motifs. . The crRNA consists of the 24-nt guide segment (G1–C24) and the 19-nt scaffold (A(19)–U(1)) (referred to as the 5' handle). The nucleotides G1–C20 in the crRNA and dC1–dG20 in the target DNA strand form the 20-bp RNA-DNA heteroduplex. The nucleotide A21 in the crRNA is flipped out and adopts a single-stranded conformation. No electron density was observed for the nucleotides A22–C24 in the crRNA and dT21–dG24 in the target DNA strand, suggesting that these regions are flexible and disordered in the crystal structure. The nucleotides dG(10)–dT(1) in the target DNA strand and dC(10*)–dA(1*) in the non-target DNA strand form a duplex structure (referred to as the PAM duplex). The . The U(1),U(16) base pair in the 5' handle is recognized by the WED domain in a base-specific manner. . The , respectively. Other representive of Cpf1 complex (Subtype V-A) from Acidaminococcus sp. BV3L6: 5kk5. from Lachnospiraceae bacterium ND2006 5id6. Subtype V-B (C2c1) Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA Class 2 CRISPR effector protein, C2c1 (classified as type V-B), has been identified to cleave DNA under the guide of crRNA:tracrRNA, distinct from a type V-A effector protein Cpf1 (type V-A, see above) that only requires a single crRNA. Furthermore, C2c1 and Cpf1 recognize different PAM sequences. Like Cpf1, C2c1 contains a conserved RuvC endonuclease domain, though it harbors a second endonuclease domain that is not well defined by sequence. C2c1 has been proved to be endonuclease-active in human cell lysates. The mechanism underlying C2c1-mediated cleavage remains elusive. The overall structure of the (5wti, from Bacillus thermoamylovorans) is a composed of an α-helical and a . The a PAM-interacting (PI) domain, a REC1 domain, a REC2 domain, and a long α helix referred to as the bridge helix (BH). The an OBD domain, a RuvC domain, and a domain with unknown functions (termed “UK” domain). The sgRNA consists of a (C1-U19), a (C(−18)-A(−24), and U(−57)-G(−61)). The guide segment and 19 nucleotides of the target DNA strand (dG(1′)-dA(19′)) form the , whereas the 9 nucleotides of the target DNA strand (dG(−1′)-dA(−9′)) and the non-target DNA strand (dC(−1*)-dT(−9*)) form a . The in the NUC lobe, composed by , interfaces with the in the REC lobe to form a . The other side of the heteroduplex is recognized by the REC2 domain. The , whereas the . The negatively charged sgRNA:target DNA heteroduplex is accommodated in the . Recognition of the sgRNA:target DNA heteroduplex by BthC2c1 is mainly through interactions between sugar-phosphate backbone and the protein. The , whereas the sugar-phosphate backbone of the target DNA sequence (dT(13′)-dA(19′)) complementary to that of PAM-distal guide segment is extensively recognized by the . The repeat:anti-repeat duplex containing an anticipated base-pairing segment (U(−6):G(−25)-G(−13):C(−18)) and an unanticipated base-pairing segment (C(−1):G(−61)-A(−5):U(−57)), is recognized by domains. The 5′-ATTC-3′ . The OBD domain consists of a β-sheet barrel flanked by four short α-helices, whereas the PI domain is composed of a bundle of four α-helices connected by linkers and loop PL1 (Ser129-Arg143). The loop PL1 deeply inserts into the minor groove of PAM duplex and interacts with the target and non-target DNA strands. from the loop PL1 hydrogen-bonds with the sugar-phosphate backbone. The sugar-phosphate backbone of PAM is recognized by via hydrogen-bonding interactions. Model of sgRNA-guided DNA cleavage by BthC2c1: --> -->

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