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.[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]
Subtype V-A (Cpf1)
Crystal Structure of Cpf1 in Complex with Guide RNA and Target DNA[4]
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 .
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[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 (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:
