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Type V CRISPR-Cas Cpf1 Endonuclease Employs a Unique Mechanism for Crrna-Mediated Target DNA Recognition

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Type V CRISPR-Cas Cpf1 Endonuclease Employs a Unique Mechanism for Crrna-Mediated Target DNA Recognition Cell Research (2016) 26:901-913. © 2016 IBCB, SIBS, CAS All rights reserved 1001-0602/16 $ 32.00 ORIGINAL ARTICLE www.nature.com/cr Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target DNA recognition Pu Gao1, 2, Hui Yang2, Kanagalaghatta R Rajashankar3, 4, Zhiwei Huang5, Dinshaw J Patel2 1Key Laboratory of Infection and Immunity, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China; 2Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA; 3Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA; 4NE-CAT, Ad- vanced Photon Source, Argonne National Laboratory, Argonne, IL 60349, USA; 5School of Life Science and Technology, Harbin Institute of Technology, Harbin 150880, China CRISPR-Cas9 and CRISPR-Cpf1 systems have been successfully harnessed for genome editing. In the CRIS- PR-Cas9 system, the preordered A-form RNA seed sequence and preformed protein PAM-interacting cleft are essential for Cas9 to form a DNA recognition-competent structure. Whether the CRISPR-Cpf1 system employs a similar mechanism for target DNA recognition remains unclear. Here, we have determined the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with crRNA and target DNA. Structural comparison between the AsCpf1-crRNA-DNA ternary complex and the recently reported Lachnospiraceae bacterium Cpf1 (LbCpf1)-crRNA binary complex identifies a unique mechanism employed by Cpf1 for target recognition. The seed sequence required for initial DNA interrogation is disordered in the Cpf1-cRNA binary complex, but becomes ordered upon ternary complex formation. Further, the PAM interacting cleft of Cpf1 undergoes an “open-to-closed” conformational change upon target DNA binding, which in turn induces structural changes within Cpf1 to accommodate the ordered A-form seed RNA segment. This unique mechanism of target recognition by Cpf1 is distinct from that reported previously for Cas9. Keywords: CRISPR-Cas; Cpf1; crRNA; genome editing Cell Research (2016) 26:901-913. doi:10.1038/cr.2016.88; published online 22 July 2016 Introduction crRNA and trans-activating crRNA (tracrRNA), or alter- natively with a synthetic single-guide RNA (sgRNA), to As one of the prokaryotic DNA sensing systems, cleave both strands of the target DNA [6-8]. A short and CRISPR-Cas (clustered regularly interspaced short palin- conserved protospacer adjacent motif (PAM) sequence dromic repeats and CRISPR-associated protein) provides near the target site is required for the cleavage process of adaptive immune protection and helps archaea and bacte- Cas9 [9, 10]. CRISPR-Cas9 has been extensively used ria defend themselves against phage infection [1-3]. De- for genome editing in various cell types and organisms pending on the architecture of the effector-CRISPR RNA [11, 12]. A series of structural studies of Streptococcus (crRNA) interference module, different CRISPR-Cas pyogenes Cas9 (SpyCas9) and its orthologs have re- systems could be assigned into two classes [1]: class 1 vealed the detailed intermolecular interactions, as well systems (multi-subunit complex, such as Cascade) [4, 5] as the conformational changes among different sub- and class 2 systems (single enzyme, such as Cas9) [6, 7]. strate-bound states [13-18]. Cas9 is the signature member of class 2 systems, which Cpf1 is a newly identified class 2 type V CRISPR-Cas functions as a multi-domain endonuclease, along with endonuclease, which has also been harnessed for genome editing in mammalian cell lines [19]. Cpf1-mediated Correspondence: Pu Gaoa, Dinshaw J Patelb cleavage is guided by a single and short (42-44 nt) crR- a E-mail: [email protected] NA [19], in contrast to Cas9 that uses both crRNA and bE-mail: [email protected] Received 6 June 2016; revised 20 June 2016; accepted 20 June 2016; pub- tracrRNA [20]. Cpf1 recognizes a T-rich PAM at the lished online 22 July 2016 5′-end of the protospacer sequence [19], in contrast to A unique mechanism for target DNA recognition of Cpf1 902 3′-G-rich PAM recognition by Cas9 [21, 22]. More im- closely related ternary complex [25], resembles a bilob- portantly, Cpf1 makes a staggered double-strand break al scaffold with an overall “Crab Claw” shape (Figure resulting in five-nucleotide 5′-overhangs distal to the 1C and 1D). AsCpf1 can be divided into two lobes: an PAM site [19], whereas Cas9 creates blunt ends proximal α-helical recognition (REC) lobe consisting of Helical-I to the PAM site [8]. Based on sequence analysis, Cpf1 and Helical-II domains, and a NUC lobe consisting of contains only one detectable RuvC endonuclease domain, OBD, LHD and RuvC domains, as well as the newly which has lead to the initial hypothesis that Cpf1 may characterized Nuc domain [25] (Figure 1A, 1C and 1D). form a dimer to cleave the two strands of target DNA [19]. The bridge helix motif is inserted between RuvC-I and Very recently, structural and functional studies show that RuvC-II motifs and connects the REC and NUC lobes Cpf1 acts as a monomer [23-25] and contains a second from the middle of the whole complex (Figure 1C). By putative novel nuclease (NUC) domain [25]. In addition comparing the individual domains of AsCpf1 with their to the target DNA interference activity, Cpf1 was also functional counterparts in SpyCas9, only the RuvC do- found to cleave precursor crRNA (pre-crRNA), leading mains show relatively good alignment (Supplementary to the generation of mature crRNAs [24]. information, Figure S1A), with a root mean square devia- Both Cas9 [26, 27] and Cpf1 [19, 24] have been tion of 4.5 Å over 145 Cα atoms, consistent with the low shown to have a seed sequence at the PAM-proximal sequence similarity outside of the RuvC domain between side of the protospacer, which is critical for DNA rec- Cpf1 and Cas9. Although an inactivating mutant protein ognition and cleavage. The 10-nt seed sequence of the (E993A) was used in this study, the overall structure of guide RNA has been shown to form a preordered A-form our ternary complex can be aligned very well with the conformation in the Cas9-sgRNA complex to facilitate recently reported structure of the AsCpf1-crRNA-DNA guide-target duplex formation [14], a mechanism that has complex that used the wild-type protein [25] (Supple- also been found in eukaryotic Argonaute complexes [28- mentary information, Figure S1B). The Y-shaped bound 30]. The Cas9-sgRNA pre-target binary conformation crRNA-DNA moiety (Figure 1E) is mostly buried within was also found to be competent for PAM recognition by the protein, with the 5′-direct repeat region of crRNA, forming a preformed PAM-interacting cleft [14]. Wheth- the crRNA-DNA heteroduplex, and the PAM-contain- er Cpf1 employs a similar strategy for target recognition ing DNA duplex binding to different surfaces within the is still unknown, although the seed segment of crRNA in AsCpf1 protein (Figure 1C and 1D). The 1:1 molar ratio Cpf1-crRNA binary complex has been predicted to most between crRNA-DNA and AsCpf1 protein indicates that likely form the A-form structure [25]. AsCpf1 acts as a monomer, in line with our size-exclu- To illuminate the molecular mechanism of substrate sion chromatography results, as well as with recently recognition of Cpf1, we determined the crystal structure of published studies [23-25]. Acidaminococcus sp. BV3L6 Cpf1 (AsCpf1) in complex with crRNA and a partially duplexed target DNA con- Intermolecular interactions between AsCpf1 and crR- taining a 5′-TTTC-3′ PAM sequence. By comparing the NA-DNA recently reported structures of pre-target-bound Cpf1-crR- The crRNA used for crystallization contains a 20-nt NA binary complex [23] with target-bound Cpf1-crRNA- direct repeat region [U(–20)-U(–1)] and a 25-nt guide DNA ternary complex (this study; see also ref. [25]), we segment (G1-C25) (Figure 1B). We initially introduced found that Cpf1 employs a unique mechanism for target one extra base pair (C25-dG1) at the end of the crR- recognition distinct from that reported for Cas9. NA-DNA heteroduplex to stabilize the nearby cleavage site, which was shown later on to have no effect based Results on structural results (see below). Most of the nucleotides were well-defined in the electron density, with the excep- Overall structure of AsCpf1E993A-crRNA-target DNA ter- tion of U(–20) and C21-C25 of crRNA, as well as dG1- nary complex dG5 of the target DNA strand (Figure 1B). Details of the We have solved the 3.29 Å crystal structure of full- intermolecular interactions in the ternary complex are length AsCpf1 carrying an inactivating mutation (E993A) summarized in Figure 2. in complex with a 45-nt crRNA, a 33-nt target DNA The 5′-direct repeat region of crRNA is bound in strand, and a 8-nt non-target DNA strand containing a the channel formed by OBD and RuvC domains (Fig- 5′-TTTC-3′ PAM sequence (Figure 1A and 1B, X-ray ure 3A). Unexpectedly, this part of the crRNA adopts statistics in Supplementary information, Table S1). The a pseudoknot fold in the complex (Figure 3A and 3B), structure of the AsCpf1-crRNA-DNA ternary complex, rather than the simple stem-loop as previously predicted which is similar to a recently reported structure of a [19]. The G(–6)-A(–2) segment forms five canonical SPRINGER NATURE | Cell Research | Vol 26 No 8 | August 2016 Pu Gao et al. 903 Figure 1 Overall structure of AsCpf1-crRNA-DNA ternary complex. (A) Domain organization of the AsCpf1 protein, together with designation of NUC and REC lobes. (B) Secondary structure diagram of crRNA (magenta) and the target DNA (black). The PAM sequence is highlighted in red.
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