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 Cpf1 complexed with the crRNA and target DNA (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 novel nuclease domain (labeled Nuc) in Cpf1 is inserted into the RuvC domain, and it is responsible for cleavage of the target strand.[1][2] 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.[3] ContentsSubtype V-A (Cpf1)Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA[4]The overall structure of the AsCpf1-crRNA-DNA complex (from Acidaminococcus sp. BV3L6, 5b43). The structure revealed that AsCpf1 adopts a bilobed architecture consisting of an α-helical recognition (REC) lobe and a nuclease (NUC) lobe, with the crRNA-target DNA heteroduplex bound to the positively charged, central channel between the two lobes. Surface representation of this scene. The REC lobe consists of the REC1 and REC2 domains, whereas the NUC lobe consists of the RuvC domain and three additional domains, denoted A, B, and C. The REC1 domain comprises 13 α-helices, and the REC2 domain comprises ten α-helices and two β-strands that form a small antiparallel sheet. Domains A and B 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. Domain C 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 WED domain is assembled from three separate regions (WED-I-III) in the Cpf1 sequence. The PI domain comprises seven α-helices and a β-hairpin. The RuvC domain contains the three motifs (RuvC-I-III) that form the endonuclease active center. A characteristic helix (referred to as the bridge helix, BH) is located between the RuvC-I and RuvC-II motifs and connects the REC and NUC lobes. The Nuc domain is inserted between the RuvC-II and RuvC-III motifs. crRNA-DNA complex. 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). Other representive of Cpf1 complex (Subtype V-A) from Acidaminococcus sp. BV3L6: 5kk5.
Subtype V-B (C2c1)Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA[5]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 BthC2c1-sgRNA-DNA ternary complex (5wti, from Bacillus thermoamylovorans) is a bi-lobed architecture composed of an α-helical recognition (REC) lobe and a nuclease (NUC) lobe. The REC lobe consists of a PAM-interacting (PI) domain, a REC1 domain, a REC2 domain, and a long α helix referred to as the bridge helix (BH). The NUC lobe contains an OBD domain, a RuvC domain, and a domain with unknown functions (termed “UK” domain). The sgRNA consists of a guide segment (C1-U19), a repeat segment (C(−1)-G(−13)), an anti-repeat segment (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 guide:target heteroduplex, 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 PAM-containing duplex (PAM duplex). The RuvC domain in the NUC lobe, composed by three split RuvC motifs (RuvC I-III), interfaces with the REC2 domain in the REC lobe to form a positively charged surface that interacts with the 3′ tail of the sgRNA. The other side of the heteroduplex is recognized by the REC2 domain. The PI domain and the N-terminal region of the REC1 domain interact with the PAM-proximal region of the heteroduplex, whereas the C-terminal regions of the REC1 and REC2 domains interact with the PAM-distal region of the heteroduplex. The negatively charged sgRNA:target DNA heteroduplex is accommodated in the positively charged channel at the interface formed by REC and NUC lobes. Recognition of the sgRNA:target DNA heteroduplex by BthC2c1 is mainly through interactions between sugar-phosphate backbone and the protein. The PAM-distal region (A13-U19) of the sgRNA interacts with the two REC domains (Lys752, Arg768, Val767, Gly765, Asp279, Tyr333, Gln323, and Lys320), 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 two REC domains (Arg769, Arg272, Thr280, Asn282, Arg294, and Arg328) and the RuvC domain (Arg841). 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 OBD (Glu412, Lys415, Leu414, Lys413, Asn452, Try451, Arg448, Arg507, and Lys9) and REC2 (Lys813, Tyr808, Lys794, Trp815, Lys793, Asn743, His783, and Asp790) domains. The 5′-ATTC-3′ PAM duplex is sandwiched between the OBD and PI domains. 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. Ser137, Lys141, and Arg140 from the loop PL1 hydrogen-bonds with the sugar-phosphate backbone. The sugar-phosphate backbone of PAM is recognized by Ser211, Val212, Ser129, Gln130, Gly132, Trp162, and Arg143 via hydrogen-bonding interactions. Model of sgRNA-guided DNA cleavage by BthC2c1:
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