Structure of the miniature type V-F CRISPR-Cas effector enzyme

•Cryo-EM structure of Cas12f in complex with a guide RNA and its target DNA•Type V-F effector consisting dimer single RNA•Molecular mechanism the RNA-guided DNA cleavage by miniature Cas12f•Evolutionary insights into possible origin type V CRISPR-Cas enzymes endonucleases derived from adaptive immune systems are widely used as powerful genome-engineering tools. Among diverse nucleases, (also known Cas14) proteins exceptionally compact associate to cleave single- double-stranded targets. Here, we report cryo-electron microscopy Cas12f1 Cas14a) DNA. Unexpectedly, revealed that two molecules assemble recognize target. Each protomer adopts different conformation plays distinct roles nucleic acid recognition cleavage, thereby explaining how enzyme achieves an “asymmetric homodimer.” Our findings augment mechanistic understanding nucleases provide framework for development tools critical therapeutic genome editing. bacteria archaea immunity against foreign acids divided classes (classes 1 2) six types (types I–VI) (Hille et al., 2018Hille F. Richter H. Wong S.P. Bratovič M. Ressel S. Charpentier E. The biology CRISPR-Cas: backward forward.Cell. 2018; 172: 1239-1259Abstract Full Text PDF PubMed Scopus (310) Google Scholar; Makarova 2020Makarova K.S. Wolf Y.I. Iranzo J. Shmakov S.A. Alkhnbashi O.S. Brouns S.J.J. Cheng D. Haft D.H. Horvath P. al.Evolutionary classification systems: burst class 2 variants.Nat. Rev. Microbiol. 2020; 18: 67-83Crossref (420) Scholar). Class include II, V, VI encompass multidomain Cas proteins, such Cas9 (type II) Cas12 V). associates dual guides (CRISPR [crRNA] trans-activating crRNA [tracrRNA]) or (sgRNA) cleaves (dsDNA) targets at sequence complementary 20 nt segment flanked NGG (where N is any nucleotide) protospacer adjacent motif (PAM) (Gasiunas 2012Gasiunas G. Barrangou R. Siksnys V. Cas9-crRNA ribonucleoprotein mediates specific bacteria.Proc. Natl. Acad. Sci. U S A. 2012; 109: E2579-E2586Crossref (1436) Jinek 2012Jinek Chylinski K. Fonfara I. Hauer Doudna J.A. A programmable dual-RNA-guided endonuclease bacterial immunity.Science. 337: 816-821Crossref (7869) (Makarova Scholar), V-A Cas12a Cpf1) binds dsDNA TTTV A, G, C) PAMs (Zetsche 2015Zetsche B. Gootenberg J.S. Abudayyeh O.O. Slaymaker I.M. Essletzbichler Volz S.E. Joung van der Oost Regev al.Cpf1 system.Cell. 2015; 163: 759-771Abstract (2055) contains nuclease domains, HNH RuvC, which strand (TS) non-TS (NTS) targets, respectively In contrast, both TS NTS RuvC domain (Swarts 2017Swarts D.C. Structural basis processing seed-dependent targeting CRISPR-Cas12a.Mol. Cell. 2017; 66: 221-233.e4Abstract (195) Swarts Jinek, 2019Swarts Mechanistic cis- trans-acting DNase activities Cas12a.Mol. 2019; 73: 589-600.e4Abstract (93) As exhibit robust eukaryotic cells, they versatile (Cong 2013Cong L. Ran F.A. Cox Lin Barretto Habib N. Hsu P.D. Wu X. Jiang W. Marraffini L.A. Zhang Multiplex engineering using CRISPR/Cas systems.Science. 2013; 339: 819-823Crossref (8854) Zetsche Recent studies identified (formerly (Harrington 2018Harrington L.B. Burstein Chen Paez-Espino Ma Witte I.P. Cofsky J.C. Kyrpides N.C. Banfield J.F. Programmed destruction CRISPR-Cas14 enzymes.Science. 362: 839-842Crossref (297) consist ∼400–700 amino residues much smaller than (∼950–1,400 acids). Cas14a1) uncultured archaeon consists 529 lacks detectable identity other except presence (Figure S1). Despite small size, crRNA:tracrRNA (which can be combined sgRNA) Scholar) TTTR R G) (Karvelis 2020Karvelis T. Bigelyte Young J.K. Hou Z. Zedaveinyte Budre Paulraj Djukanovic Gasior Silanskas al.PAM CRISPR-Cas12f triggers cleavage.Nucleic Acids Res. 48: 5016-5023Crossref (47) similarity those enzymes, Yamano 2016Yamano Nishimasu Hirano Li Y. Fedorova Nakane Koonin E.V. al.Crystal Cpf1 DNA.Cell. 2016; 165: 949-962Abstract (320) Cas12b (Shmakov 2015Shmakov Semenova Minakhin Konermann Severinov al.Discovery functional characterization systems.Mol. 60: 385-397Abstract (577) Yang 2016Yang Gao Rajashankar K.R. Patel D.J. PAM-dependent C2c1 endonuclease.Cell. 167: 1814-1828.e12Abstract (114) Cas12e CasX) (Burstein 2017Burstein Harrington Strutt S.C. Probst A.J. Anantharaman Thomas B.C. New uncultivated microbes.Nature. 542: 237-241Crossref (264) Liu 2019Liu J.J. Orlova Oakes B.L. Spinner H.B. Baney K.L.M. Chuck Tan Knott G.J. al.CasX comprise family genome.Nature. 566: 218-223Crossref (170) Therefore, action remains enigmatic. We examined vitro activity purified (hereafter referred simplicity) sgRNA toward TTTG PAM. efficiently cleaved 25–50 mM but not 100–150 NaCl concentrations (Figures S2A S2B), consistent previous study addition, D326A mutant did DNA, previously observed Karvelis confirming D326 S2C). To clarify Cas12f-mediated mechanism, determined cryoelectron (cryo-EM) (the inactive mutant) (180 nt) (40 bp) PAM, overall resolution 3.3 Å 1A–1D S3A–S3H; Video S1; Table 1). Surprisingly, (referred Cas12f.1 Cas12f.2) one molecule form 1A–1D). amino-terminal (NTD) carboxy-terminal (CTD), connected linker loop. NTD three domains: wedge (WED), (REC), zinc finger (ZF) domains. CTD another ZF domain, termed acid-binding (TNB) domain. bilobed architecture REC lobe (NUC) lobe, RNA-target heteroduplex bound central channel between lobes 1B 1C). formed WED, ZF, domains (WED.1/ZF.1/REC.1) Cas12f.2 (WED.2/ZF.2/REC.2), whereas NUC TNB (RuvC.1/TNB.1) (RuvC.2/TNB.2).Table 1Cryo-EM data collection refinement statisticsData processingSampleCas12f-sgRNA-DNAEMDB IDEMD-30299PDB ID7C7LMicroscopeTitan Krios G3iDetectorGatan K3 cameraMagnification105,000Voltage (kV)300Electron exposure (e−/Å2)48.7Defocus range (μm)−0.8 −1.6Pixel size (Å/px)0.83Symmetry imposedC1Number movies2,848Initial particle images1,960,343Final images87,253Map (Å)3.3FSC threshold0.143Map-sharpening B factor (Å2)−107.977Model building refinementModel compositionProtein atoms6,828Nucleic atoms3,317Metals3RMSDs idealBond lengths (Å)0.0074Bond angles (°)1.498ValidationClashscore5.35Rotamer outliers (%)8.05Ramachandran plotFavored (%)90.5Allowed (%)9.1Outlier (%)0.5 Open table new tab https://www.cell.com/cms/asset/5d8261e8-40ad-4ef3-8eba-a4fc0beb5e24/mmc2.mp4Loading ... Download .mp4 (110.76 MB) Help files S1. Structure Cas12f-guide complex, related Figure WED comprises seven-stranded β-barrel α helix β hairpin oligonucleotide/oligosaccharide-binding (OB) fold similar (Yamano Stella 2017Stella Alcón Montoya R-loop after cleavage.Nature. 546: 559-563Crossref (86) despite their limited S4A). inserted strands β1 β2 CCCH-type ion coordinated C50, H53, C69, C72 S4A S4B). four helices thus contributes mainly miniaturization has RNase H fold, five-stranded mixed sheet helices, D326, E422, D510 catalytic center β5 α6 CCCC-type C475, C478, C500, C503 S4C). cysteine conserved among An X-ray fluorescence elemental analysis protein indicated ions S4D). Nuc target-strand loading [TSL] Cas12e) adopt structures contain CXXC motifs, while Cas12e. have unrelated facilitate (Yang described these probably participate placement active site. propose should collectively structural comparison notable difference arrangement CTD, facilitated flexible loop local variations individual S5A–S5F). ZF.1 (residues 18–93), WED.1 256–286), RuvC.1 368–382) interact scaffold, equivalent regions exposed solvent disordered 1B–1D S5A–S5F), indicating undergoes changes upon binding. (U[−160]–C20) 160 comprising five stems (stems 1–5) pseudoknot (PK) 2A, 2B, S6A, S6B). Stem (U[−160]–A[−141]) upper stem (A[−129]–U[−103]) 5 (A[−29]–G[−13]) structure, suggesting flexibility regions. unexpected features scaffold. First, U(−84)–U(−79) base pair A(−7)–A(−2) PK (crRNA repeat-tracrRNA anti-repeat duplex [R:AR-1]). coaxially stacks 3 continuous helix. Second, G(−13)–A(−11) do predicted C(−26)–U(−28) A(−12), A(−11), G(−10) flipped out stem, A(−29)–C(−26), rather A(−25)–U(−22), U(−14)–G(−17) complete (R:AR-2). Indeed, deletion A(−25)–U(−22) affect S6C). Third, triples, G(−89)–C(−75)⋅A(−33) (stem 3), G(−64)–C(−39)⋅A(−62) 4a), U(−60)–A(−42)⋅A(−43) 4b), stabilize scaffold S6D). dimerizes through interfaces asymmetric manner 3A). primary interface symmetric hydrophobic residues, I118, Y122, I126, M178, REC.1 REC.2 3B). secondary α1-α2 α1 α2 RuvC.2 3C). H371.1 N369.1 hydrogen bond C405.2/D409.2 R402.2, respectively, L365.1 interacts S347.2, N349.2, D350.2. I118R, Y122A, I126R, M178R mutations reduced 3D, S7A, S7B). Furthermore, I118R/Y122A/I126R/M178R (RARR) lacked 3D). sgRNA, wild-type (WT) RARR eluted similarly size-exclusion column position corresponding molecular mass 198 kDa, (Cas12f)2-sgRNA (184 kDa), Cas12f-sgRNA (121 kDa) S7C). These WT least under tested conditions. Together, our suggested correct positioning relative (i.e., integrity interface) required (in particular, formation), formation. analyzed oligomeric states chromatography. control, created dimeric (Dimer), S7A absence later Dimer S7D), dimerization. extensively recognized Cas12f.2, playing 4 5A–5F ). interactions lower region 5C). C(−140)–G(−91) basal F359.2 A360.2. G(−138) A(−100) sandwiched K330.2 F352.2 bonds D348.2/R438.2 K330.2, respectively. (U[−160]–G[−94]), (U[−160]–A[−144]), 5G), importance 2. 3-PK WED.1, ZF.1, sugar-phosphate backbone 4, 5A, 5B). first U(−84)–A(−2) N262.1 K398.1 bonds, U(−84) A(−2) stacking R259.1 C(−1), 5D). C(−1) strictly R259.1, T271.1, E272.1. U(−92)⋅U(−73) A360.2, R361.2, I364.2 bridges 4a) 5E). nucleobases C(−39), G(−66)–C(−37), A(−35) 4a G375.1, H376.1, K383.1 loop, Notably, C(−40) side chains A378.1, K381.1, L382.1 main K367.1, G377.1 5E) participates RNA-DNA recognition. 366–383) abolished S7A). results interaction cleavage. 4b 4c) charge shape complementarity W95.1/K299.1, Y82.1/W95.1, V15.1/L253.1/Q257.1, A(−11) forming W95.1 5F). syn forms multiple D213.1, S255.1, T256.1. 39–72) impaired ZF.2 S7A), RNA. accommodated within positively charged 5B), RNA-dependent Cas12f. seven nucleotides (dG1∗–dT7∗) displaced, single-stranded REC.2/WED.2 sequence-independent manner. H139.1, I131.1/Y232.2, P234.2 dG1∗, dA3∗, dA5∗ NTS, N133.1, K173.1, R103.2, R292.2 5H). observe clear densities (−8)–(−1) 28–32 TS, (−12∗)–(−8∗) 8∗–28∗ flexibility. PAM-containing 5I). dT(−4∗)–dT(−2∗) PAM A156.1 Y146.1. Y146.1 also phosphate group dT(−4∗) dT(−3∗). nucleobase dG(−1∗) hydrogen-bonding S142.1 R163.1, dA24 dA23, dT(−3∗), Y202.1 Q197.1, Y146A Q197A activities, contact Q197.1 observations explain preference dC21 dC20 K198.1 S286.1 (K198.2 S286.2 disordered) 5I facilitating formation, 221-233.e4