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]Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. The crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.8 A˚ resolution was reported. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences, thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif (PAM) by base and shape readout mechanisms. These findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox. The microbial adaptive immune system CRISPR-Cas helps bacteria and archaea defend themselves against the invasion of foreign nucleic acids. The CRISPR-Cas systems encompass arrays of direct repeats that are separated by unique spacers derived from foreign DNA. The repeat arrays are transcribed into long transcripts (precursors of CRISPR RNAs), which are then processed to yield small CRISPR RNAs (crRNAs), consisting of a spacer and a portion of the adjacent direct repeat. The crRNAs form a complex with Cas endonucleases, and in some cases with accessory Cas proteins as well, and serve as guidesto target and cleave the cognate foreign nucleic acid, thus achieving interference. DNA recognition by Cas-crRNA complexes requires the presence of a protospacer adjacent motif (PAM) near the target site, which contributes to self versus non-self discrimination. Recently, a second class 2 (type V) effector protein, Cpf1, has been harnessed for genome editing. Similar to Cas9, Cpf1 can be reprogrammed to target DNA sites of interest through complementarity to a guide RNA. However, Cpf1 possesses several unique features that distinguish it from Cas9 and could provide for a substantial expansion of the genome editing toolbox. First, Cpf1 is guided by a single crRNA, whereas Cas9 uses a crRNA and a second small RNA species, a transactivating crRNA (tracrRNA). Second, Cpf1 recognizes a T-rich PAM, in contrast to the G-rich PAM favored by Cas9. Third, Cpf1 generates staggered ends in its PAM-distal target site, whereas Cas9 creates blunt ends within the PAM-proximal target site. Fourth, Cpf1 contains the RuvC domain but lacks a detectable second endonuclease domain, whereas Cas9 uses the HNH and RuvC endonuclease domains to cleave the target and non-target DNA strands, respectively. Together, these observations imply major differences in the target DNA recognition and cleavage mechanisms between Cas9 and Cpf1. To clarify how Cpf1 recognizes and cleaves DNA targets, the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the crRNA and its double-stranded DNA target containing the 5'-TTTN-3' PAM was determined. AsCpf1 adopts a bilobed architecture that accommodates the crRNA-target DNA heteroduplex in the central channel. AsCpf1 recognizes the crRNA scaffold and the 5'-TTTN-3' PAM in structure- and sequence-dependent manners. AsCpf1 contains a RuvC endonuclease domain and a putative novel nuclease domain, which are located at positions suitable to induce staggered DNA double-strand breaks. The structural comparison of AsCpf1 with Cas9 reveals both striking structural similarity and substantial differences between the two class 2 effector proteins, thus explaining their distinct functionalities and suggesting their functional convergence. Overall Structure of the AsCpf1-crRNA-Target DNA Complex 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. Structure of the crRNA and Target DNA 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). Recognition of the 5' Handle of the crRNA The 5' handle of the crRNA is bound at the groove between the WED and RuvC domains. The U(1),U(16) base pair in the 5' handle is recognized by the WED domain in a base-specific manner. U(1) and U(16) hydrogen bond with His761 and Arg18/Asn759, respectively, while U(1) stacks on His761. The N6 of A(19) hydrogen bonds with Leu807 and Asn808, while the base moieties of A(18) and A(19) form stacking interactions with Ile858 and Met806, respectively. Recognition of the crRNA-Target DNA Heteroduplex The crRNA-target DNA heteroduplex is accommodated within the positively charged, central channel formed by the REC1, REC2, and RuvC domains and is recognized by the protein in a sequence-independent manner. The PAM-distal and PAM-proximal regions of the heteroduplex are recognized by the REC1-REC2 domains and the WED-REC1-RuvC domains, respectively. Recognition of the target DNA strand by the the RuvC domain. Arg951 and Arg955 in the bridge helix, which interact with the sugar-phosphate backbone of the target DNA strand, are conserved among the Cpf1 family members. Notably, the sugar-phosphate backbone of the nucleotides G1–A8 in the crRNA forms multiple contacts with the WED and REC1 domains, and the base pairing within the 5-bp PAM-proximal ‘‘seed’’ region is important for Cpf1-mediated DNA cleavage. The side chain of Trp382 in the REC2 domain forms a stacking interaction with the C20:dG20 base pair in the heteroduplex and thus prevents base pairing between A21 and dT21. Recognition of the 5'-TTTN-3' PAM The PAM duplex adopts a distorted conformation with a narrow minor groove, as often observed in AT-rich DNA, and is bound to the groove formed by the WED, REC1, and PI domains. The PAM duplex is recognized by the WED-REC1 and PI domains from the major and minor groove sides, respectively. Lys607 in the PI domain is inserted into the narrow minor groove and plays critical roles in the PAM recognition. The O2 of dT(2*) forms a hydrogen bond with the side chain of Lys607, whereas the nucleobase and deoxyribose moieties of dA(2) form van der Waals interactions with the side chains of Lys607 and Pro599/Met604, respectively. Structural observations can explain the requirement of the third T in the 5'-TTTN-3' PAM. The 5-methyl group of dT(3*) forms a van der Waals interaction with the side-chain methyl group of Thr167, whereas the N3 and N7 of dA(3) form hydrogen bonds with Lys607 and Lys548, respectively. The 5-methyl group of dT(4*) is surrounded by the side-chain methyl groups of Thr167 and Thr539, whereas the O4' of dA(4) forms a hydrogen bond with the side chain of Lys607. Notably, the N3 and O4 of dT(4*) form hydrogen bonds with the N1 of dA(4) and the N6 of dA(3), respectively. Together, these results demonstrate that AsCpf1 recognizes the 5'-TTTN-3' PAM via a combination of base and shape readout mechanisms. Thr167 and Lys607 are conserved throughout the Cpf1 family, and Lys548, Pro599, and Met604 are partially conserved. These observations indicate that the Cpf1 homologs from diverse bacteria recognize their T-rich PAMs in similar manners, although the fine details of the interaction could vary. The RuvC-like Endonuclease and a Putative Second Nuclease Domain The RuvC domain comprises a typical RNase H fold, consisting of a five-stranded mixed β-sheet (β1–β5) flanked by three α-helices (α1–α3), and two additional α-helices and three β-strands. The conserved, negatively charged residues Asp908, Glu993, and Asp1263 form an active site. Notably, the bridge helix is inserted between strand β3 and helix α1 in the RNase H fold and interacts with the REC2 domain. The main-chain carbonyl group of Gln956 in the bridge helix forms a hydrogen bond with the side chain of Lys468 in the REC2 domain. In addition, Trp958 in the RuvC domain is accommodated in the hydrophobic pocket formed by Leu467, Leu471, Tyr514, Arg518, Ala521, and Thr522 in the REC2 domain. These residues, with the exceptions of Leu467 and Ala521, are highly conserved among the Cpf1 family members, and the W958A mutant exhibited reduced activity. These observations highlight the functional importance of the bridge helix-mediated interaction between the REC and NUC lobes. The crystal structure revealed the presence of the Nuc domain, which is inserted between the RuvC-II (strand β5) and RuvC-III (helix α3) motifs in the RuvC domain. The Nuc domain is connected to the RuvC domain via two linker loops (referred to as L1 and L2). The Nuc domain comprises five α-helices and nine β-strands and lacks detectable structural or sequence similarity to any known nucleases or proteins. Notably, the conserved polar residues Arg1226 and Asp1235 and the partially conserved Ser1228 are clustered in the proximity of the active site of the RuvC domain. The S1228A mutant showed DNA cleavage activity comparable to that of wild-type AsCpf1. In contrast, the D1235A mutant exhibited reduced activity, whereas the R1226A mutant showed almost no activity, indicating that Arg1226 is critical for DNA cleavage. Further characterization revealed that the R1226A mutant acts as a nickase that cleaves the non-target DNA strand, but not the target strand, indicating that the Nuc and RuvC domains cleave the target and non-target DNA strands, respectively. The mutations of the catalytic residues in the AsCpf1 RuvC domain abolished the cleavage of both DNA strands, suggesting that the cleavage of the non-target strand by the RuvC domain is a prerequisite for the target strand cleavage by the Nuc domain, presumably via a conformational change in the complex.
Other representatives of Cpf1 complex (Subtype V-A)
Subtype V-B (C2c1)Structural basis of stringent PAM recognition by CRISPR-C2c1 in complex with sgRNA[7]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. To map the DNA cleavage site of BthC2c1, Sanger sequencing was performed to analyze the DNA ends of the cleaved products of in vitro cleavage reactionsWe found that BthC2c1-cleaved DNA products had a 7-nt 5′ overhang, differing from the blunt DNA cleavage mode of Cas9. This staggered double-stranded cleavage occurred after the 16th nucleotide on the non-target strand and after the 23rd nucleotide on the target strand distal to the PAM sequence. The BthC2c1 cleavage site on the target strand is located outside the guide:target heteroduplex segment. This is distinct from Cas9 and Cpf1 (see above), both of which cleave the target strand within the guide:target heteroduplex segment. Interestingly, the target strand cleavage mode of BthC2c1 resembles that of C2c2 (CRISPR type VI), although C2c2 digests crRNA-guided RNA substrates. Other representatives of C2c1 complex (Subtype V-B)
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