Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4Ruchao Penga,b,1, Zhiteng Lia,1, Ying Xuc,1, Shaoshuai Hea, Qi Pengb, Lian-ao Wud, Ying Wue, Jianxun Qia,b, Peiyi Wangf,Yi Shia,b,g,h,2, and George F. Gaoa,b,g,h,i,2

aSavaid Medical School, University of Chinese Academy of Sciences, 100049 Beijing, China; bChinese Academy of Sciences (CAS) Key Laboratory ofPathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, 100101 Beijing, China; cSchool of Life Sciences,University of Science and Technology of China, 230026 Hefei, China; dInstitute of Physical Science and Information Technology, Anhui University, 230039Hefei, China; eSchool of Basic Medical Sciences, Wuhan University, 430071 Wuhan, China; fDepartment of Biology, Southern University of Science andTechnology, 518055 Shenzhen, China; gCenter for Influenza Research and Early-Warning, Chinese Academy of Sciences, 100101 Beijing, China; hShenzhenKey Laboratory of Pathogen and Immunity, Shenzhen Third People’s Hospital, 518112 Shenzhen, China; and iNational Institute for Viral Disease Control andPrevention, Chinese Center for Disease Control and Prevention, 102206 Beijing, China

Edited by Emmanuelle Charpentier, Max Planck Unit for the Science of Pathogens, Berlin, Germany, and approved August 7, 2019 (received for review May31, 2019)

Prokaryotes possess CRISPR-Cas systems to exclude parasitic pred-ators, such as phages and mobile genetic elements (MGEs). Thesepredators, in turn, encode anti-CRISPR (Acr) proteins to evade theCRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibitingthe CRISPR-Cas12a system, was shown to diminish Lachnospiraceaebacterium Cas12a (LbCas12a)-mediated genome editing in humancells, but the underlying mechanisms remain elusive. Here we reportthe cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at sev-eral stages of the CRISPR-Cas working pathway, different fromother characterized type I/II Acr inhibitors which target only 1 stage.First, it locks the conformation of the LbCas12a-crRNA complex toprevent target DNA-crRNA hybridization. Second, it interacts withthe LbCas12a-crRNA-dsDNA complex to release the bound DNA be-fore cleavage. Third, AcrVA4 binds the postcleavage LbCas12a com-plex to possibly block enzyme recycling. These findings highlight themultifunctionality of AcrVA4 and provide clues for developing reg-ulatory genome-editing tools.

CRISPR-Cas system | anti-CRISPR proteins | Cas12a | AcrVA4 |inhibition mechanism

Prokaryotes utilize a panel of defense systems to fight againstthe parasitic predators, including phages and mobile genetic

elements (MGEs) (1). In addition to the innate immune systems,such as the restriction-modification (R-M) system (2), the toxin-antitoxin abortive infection system (3), DNA interference (4), thebacteriophage exclusion (BREX) system (5), etc., they have alsodeveloped a highly specific and prevalent adaptive immune systemknown as the CRISPR-Cas (clustered regularly interspaced shortpalindromic repeats [CRISPR]-associated proteins) system (6). Ithas been estimated that almost all archaea and about 50% ofbacteria encode CRISPR-Cas systems in their genomes (7).CRISPR-Cas systems can protect the prokaryotes against phageinfection by targeting the foreign nucleic acids in a sequence-specific manner, which works through 3 stages (adaptation, bio-genesis, and interference) in the process (8, 9). Initially, the for-eign nucleic acid segments are acquired by the host bacteria andincorporated into the CRISPR array as spacers in their genomes.These sequences are transcribed and processed into matureCRISPR RNA (crRNA) that contains a short direct repeat and aspacer. The Cas proteins are then expressed and associate withcrRNA to form ribonucleoprotein complexes which detect theforeign nucleic acids by recognizing the protospacer adjacentmotif (PAM), followed by base pairing between the spacer and thetarget sequence, and finally destroy the foreign nucleic acids (8).CRISPR-Cas systems fall into 2 classes (I and II) and are

further classified into 6 types (I–VI) so far, based on their phy-logeny and working mechanisms (10, 11). All class I CRISPR-Cas systems, including types I, III, and IV, utilize a multisubunit

effector complex, known as the surveillance complex, for targetrecognition and perform subsequent cleavage by recruiting anadditional Cas protein or with resident nuclease subunits in thecomplex. By contrast, the class II systems, including type II, V, andVI, encode a single multidomain Cas protein to form the effectorcomplex with crRNA, which mediates both the target recognitionand degradation processes (10, 11). Due to the high sequencespecificity and simple effector composition, 2 class II CRISPR-Caseffectors, Cas9 and Cas12a (Cpf1), have been successfully har-nessed as powerful tools for genome editing, providing tremen-dous promise for therapeutic applications (12, 13). Another newlyidentified Cas13a (C2c2), which cleaves RNA targets, has alsobeen developed as a rapid and high-sensitivity detection tool fordiagnosis of pathogens and genotyping (14). Cas12a is guided by asingle crRNA and generates staggered ends in its PAM-distal targetsite (15), in contrast to the 2-RNA-guided Cas9 enzyme whichgenerates blunt ends at the cleavage site (16). As a next-generation

Significance

Bacteriophage encodes anti-CRISPR (Acr) proteins to inactivatehost bacterial CRISPR-Cas systems. These Acrs are also found inbacteria to avoid self-targeting autoimmunity. So far, quite afew Acrs targeting type I and II CRISPR-Cas systems have beenwell characterized. In contrast, the Acrs inhibiting type V systemsremain poorly understood. Both type II (Cas9) and V (Cas12a)CRISPR-Cas systems have been harnessed as powerful tools forgenome editing and the latter showed even better efficiencyand accuracy. In this work, we report a comprehensive mecha-nistic insight into a unique multistage inhibitor, AcrVA4, blockingCRISPR-Cas12a activity, different from other characterized single-stage targeting Acrs. This represents a sophisticated mechanismfor CRISPR-Cas inhibition and provides clues for developingregulatory tools for genome editing.

Author contributions: R.P., Y.S., and G.F.G. designed research; R.P., Z.L., Y.X., S.H., Q.P.,L.W., Y.W., J.Q., and P.W. performed research; R.P., Z.L., Y.X., S.H., J.Q., Y.S., and G.F.G.analyzed data; and R.P., Y.S., and G.F.G. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The cryo-EM density maps of form A and form B LbCas12a-crRNA-AcrVA4 complexes have been deposited to the Electron Microscopy Data Bank (EMDB)with the accession codes EMD-0704 and EMD-0705, respectively. The coordinates of cor-responding atomic models have been deposited to the Protein Data Bank (PDB) under theentries 6KL9 and 6KLB, respectively.1R.P., Z.L., and Y.X. contributed equally to this work.2To whom correspondence may be addressed. Email: shiyi@im.ac.cn or gaof@im.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1909400116/-/DCSupplemental.

Published online August 29, 2019.

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genome-editing tool, Cas12a can simultaneously manipulate mul-tiple target genes with potentially higher precision as compared toCas9 (13, 17).On the other hand, phages and MGEs, in turn, can encode

anti-CRISPR (Acr) proteins to inactivate the CRISPR-Cas sys-tems in host bacteria (18, 19). To date, a panel of Acr proteinshave been discovered for type I, II, and V systems, through iso-lation of CRISPR-resistant phages, or by proximity to anti-CRISPR-associated (aca) genes, or by screening of bacteria ge-nomes with self-targeting spacer sequences (20–29). A total of5 type V Acrs have been identified and only 3 (AcrVA1, AcrVA4,and AcrVA5) of them can inhibit CRISPR-Cas12a-mediated ge-nome editing in human cells, among which AcrVA4 showed themost potent inhibition activity for Lachnospiraceae bacteriumCas12a (LbCas12a) (28, 29). Recently, Dong et al. reportedAcrVA5 inhibits Cas12a activity by acetylation, which renderssteric hindrance for PAM recognition to inhibit dsDNA binding(30). Knott et al. found AcrVA1 enables the degradation of spacersequence in the Cas12a-crRNA complex and AcrVA4 interfereswith dsDNA binding by biochemical characterization (31). How-ever, the exact underlying mechanisms for AcrVA4-mediatedCas12a inhibition remain unclear.In this study, we report the structural basis of AcrVA4 inhibiting

LbCas12a-mediated DNA cleavage. We found that AcrVA4 canbind to both crRNA-loaded LbCas12a and LbCas12a-crRNA-dsDNA complex, but not the apo form, with ultrahigh bindingaffinity. AcrVA4 can lock the conformation of LbCas12a-crRNAcomplex (crRNA-loaded state) to block target DNA hybridizationwith the crRNA spacer. It can also interact with the LbCas12a-crRNA-dsDNA complex (full R-loop conformation) to release thebound DNA before cleavage. In addition, it binds the postcleavageR-loop complex to block the recycling usage of the enzyme. Thesefindings expand our understanding on the diverse molecularmechanisms of Acr proteins silencing CRISPR-Cas immunity andoffer guidelines for developing anti-CRISPR off-switch tools forgenome engineering and related biotechnological applications.

ResultsAcrVA4 Homodimer Directly Interacts with Both LbCas12a-crRNAand LbCas12a-crRNA-dsDNA Complexes. Previous study has shownthat AcrVA4 could efficiently inhibit LbCas12a-mediated dsDNAcleavage both in vitro and in vivo (28, 29). To investigate whetherAcrVA4 inactivates LbCas12a by direct interaction, we individuallyexpressed and purified AcrVA4 and LbCas12a proteins using theEscherichia coli (E. coli) expression system and tested their bindingby biochemical studies. As shown by size-exclusion chromatogra-phy (SEC) assay, AcrVA4 was eluted as a monodispersed peakwith an estimated molecular weight (MW) of ∼50 kDa, indicatinga dimeric form in solution (Fig. 1A). SDS-PAGE analysis underreducing and nonreducing conditions revealed the presence ofinterchain disulfide (Fig. 1B). Further analytical ultracentrifuga-tion assay showed the MW of soluble AcrVA4 is ∼54 kDa, con-firming that AcrVA4 exists as a homodimer cross-linked byinterchain disulfide (Fig. 1C).It has been established that significant conformational

changes occur during the transitions from apo Cas12a to crRNA-loaded and dsDNA target-bound states (32–36). We then per-formed SEC assays to detect the potential interactions betweenAcrVA4 and LbCas12a at these different conformational states.The apo LbCas12a showed no binding to AcrVA4 (Fig. 1D),whereas the LbCas12a-crRNA complex efficiently interacted withAcrVA4 and formed a stable complex in solution (Fig. 1E).Strikingly, AcrVA4 could also bind to the LbCas12a-crRNA-dsDNA complex in full R-loop conformation, using catalyticallydead LbCas12a mutant with an E925Q substitution (dLbCas12a),and induce the release of bound dsDNA to avoid cleavage (Fig. 1Fand SI Appendix, Fig. S1 A and B). This interaction could possiblyrevert the conformation of LbCas12a back to the crRNA-loaded

state to deactivate the enzyme. In addition, AcrVA4 could alsointeract with the postcleavage R-loop complex but the boundDNA did not dissociate (SI Appendix, Fig. S1 C and D). This in-teraction would probably prevent the new crRNA replacementprocess for Cas12a resetting (32), which therefore blocks the en-zyme recycling for next-round catalysis. These evidences suggestAcrVA4 could potentially inhibit the activity of Cas12a at severalstates, both before and after dsDNA binding.

Structural Features of AcrVA4. To elucidate the mechanism ofAcrVA4-mediated Cas12a inhibition, we determined the structureof AcrVA4 bound to the LbCas12a-crRNA complex at 3.25-Åresolution by cryo-electron microscopy (cryo-EM) single particlereconstruction (SI Appendix, Figs. S2–S4). This structure corrob-orated our biochemical evidence that AcrVA4 exists in dimericform with an interchain disulfide (Fig. 1G). The structure ofAcrVA4 is composed of 2 domains, the N-terminal and C-terminaldomains (NTD and CTD), connected by a central helix (Fig. 1 Gand H). Two AcrVA4 protomers form a dumbbell-shaped struc-ture with a near-parallel orientation in which the central helicesstabilize the dimeric interactions. The 2 central helices are notstrictly parallel to each other but form a crossover in the middlewhere an interchain disulfide is observed between C131 and itssymmetric partner (Fig. 1G). Apart from the disulfide, the dimericinterface is further stabilized by quite a few polar and nonpolarinteractions between the 2 helices (Fig. 1G). To test the role ofinterchain disulfide for AcrVA4 dimerization, we conducted SECand sedimentation analyses under reducing conditions and foundthe dimerization interactions were well maintained (SI Appendix,Fig. S5A). Moreover, the SEC profiles for binding the LbCas12a-crRNA complex were essentially the same in the presence or ab-sence of dithiothreitol (DTT) (SI Appendix, Fig. S5B). Besides, theC131S mutant also displayed homogeneous dimeric form in solu-tion (SI Appendix, Fig. S5C). These observations demonstrated thatthe interchain disulfide is not essential for AcrVA4 dimerization.The CTD of AcrVA4 is mainly composed of 6 β-strands con-

nected by flexible loops in between (Fig. 1H) and is responsible forCas12a interacting (Fig. 2). The NTD of AcrVA4 is located at thedistal end of the complex and could not be clearly resolved due topoor density (Fig. 1G). Based on secondary structure predictions,the NTD of AcrVA4 might form a stable domain with orderedstructure (SI Appendix, Fig. S5D). The failure of reconstructingthis region indicated the connecting loop between NTD and thecentral helix rendered the entire domain highly flexible. We thenperformed a DALI (37) search to compare the structure ofAcrVA4 with other known protein structures, but no signifi-cant hint was obtained, indicating this structure represents anovel fold and further supporting the diverse origins of differentAcrs in evolution.

AcrVA4 Binds LbCas12a-crRNA with Different Stoichiometries.Duringcryo-EM image processing, we noticed the LbCas12a-crRNA-AcrVA4 complex exists in 2 forms with different binding stoichi-ometries. The predominant form (form A, ∼90% of all particles)contains an AcrVA4 homodimer and a single copy of theLbCas12a-crRNA complex, while the minor form (form B, ∼10%of all particles) contains 2 copies of LbCas12a-crRNA binding toan AcrVA4 dimer (Fig. 2C and SI Appendix, Fig. S2 B and C).This observation was consistent with the SEC profile of theLbCas12a-crRNA-AcrVA4 complex that was eluted as a domi-nant peak with a small shoulder in the front, corresponding toform A and form B complexes, respectively (Fig. 1E). The2 complex forms were reconstructed at 3.25-Å and 4.10-Å reso-lutions, respectively (SI Appendix, Fig. S3A and Table S1).The binding of AcrVA4 to LbCas12a is mediated by its CTD

that inserts into a valley formed by the wedge (WED), bridge helix(BH), REC2, and RuvC domains of LbCas12a and the crRNA(Fig. 2 A and B). The interactions mainly involve 1 protomer

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(AcrVA4.1) within the AcrVA4 dimer and the other protomer(AcrVA4.2) contributes few Van der Waals (VDW) contacts (Fig.2B and SI Appendix, Table S2). In the form A complex, the vacantCas12a binding site on AcrVA4.2 is fully exposed and thus offeringthe opportunity for the binding of a second Cas12a molecule (Fig.2B). As observed in the form B complex, 2 copies of the LbCas12a-crRNA complex are cross-linked by an AcrVA4 homodimer toform a near-symmetric structure (Fig. 2D and SI Appendix, Fig.S4D), which corroborated the binding interface identified in theform A complex.Both complexes display highly similar conformations for

interacting with AcrVA4, indicating a common mechanism forCas12a inhibition (SI Appendix, Fig. S3B). This observation alsoimplies that the low abundance of the form B complex comparedto form A might result from the excessive amount of AcrVA4in the preparation. To test this hypothesis, we incubated theLbCas12a-crRNA complex with AcrVA4 at a molar ratio of 2:1(2 LbCas12a-crRNA complexes versus 1 AcrVA4 dimer) andfound that most of the complex exists in form B stoichiometry (SIAppendix, Fig. S5E), in contrast to the form A dominant profilewhen an excessive amount of AcrVA4 is provided. This evidence

suggests that both form A and B binding modes are equiva-lently functional for Cas12a inhibition, which might be prefer-entially adopted in response to different AcrVA4 concentrationsin the cell.

Interactions between AcrVA4 and the LbCas12a-crRNA Complex. Thebinding interface between AcrVA4 and the LbCas12a-crRNAcomplex could be divided into 3 subregions. The C-D loop ofAcrVA4 forms a long protrusion stretching into the valley sur-rounded by the WED, BH, and REC2 domains of LbCas12a (Fig.3 A and B). At the bottom of the valley, the tip of the C-D loopalso contacts with the crRNA repeat and the RuvC domain. TheB/C strands and A-B/E-F loops form 2 wings to cover the valley onboth sides (Fig. 3B). The other AcrVA4 protomer (AcrVA4.2)within the homodimer forms several VDW contacts with theWED domain outside the valley (Fig. 3A and SI Appendix, TableS2), contributing almost no effect to the interaction.The B/C strands (residues 183 through 196) contact with the

WED domain of LbCas12a mainly through polar interactions(Fig. 3C). Residue E184 within the B strand is interspaced be-tween H759 and K768 of LbCas12a, forming 2 consecutive salt

Fig. 1. AcrVA4 forms a homodimer and selectively binds the LbCas12a-crRNA binary complex. (A) SEC of AcrVA4 using a Superdex 200 10/300 GL column. (B)SDS-PAGE profiles of AcrVA4 at reduced and nonreduced conditions. The bands of monomeric and dimeric forms are indicated by black and red triangles,respectively. (C) Sedimentation velocity analysis of AcrVA4. The estimated MW corresponds to a homodimer. (D–F) Analytical SEC assays for testing thebinding of AcrVA4 to apo LbCas12a (D), LbCas12a-crRNA binary complex (E), and dLbCas12a-crRNA-dsDNA ternary complex (F). (G) EM density and atomicstructure of the AcrVA4 dimer. One of the protomers is colored by domains and the other is colored in gray. The interchain disulfide is highlighted by a reddashed oval. The dimeric interface is shown in close-up view and the interacting residues are shown as sticks (colored by atoms). Hydrogen bonds and saltbridges are represented as black dashed lines. (H) Topological diagram of the AcrVA4 structure. As most of the NTD could not be resolved in the density map,it is represented by a dashed oval (cyan).

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bridges in the interface. The main chain of R187 in the B strand isconnected to the main chain of V758 in LbCas12a by 2 hydrogenbonds. Besides, residue M186 of the B strand forms extensiveVDW interactions with V758 and H759 of LbCas12a, as well asthe nucleotide A-20 within the CRISPR repeat of crRNA. Inaddition, the connecting loop between the central helix and CTDof AcrVA4 also participates in the interaction (Fig. 3C).The C-D loop (residues 197 through 208) of AcrVA4 protrudes

deep inside the valley and interacts with multiple domains (REC2,WED, and RuvC) of LbCas12a, as well as the crRNA repeat (Fig.3B). This loop contains quite a few charged residues, formingextensive electrostatic interactions with both LbCas12a and thephosphate of crRNA. The main chain oxygen of K202 hydro-gen bonds to the side chain of N895 in the RuvC domain, andits side chain forms a salt bridge with the phosphate of U-14 inthe crRNA. Residue R203, E204, and R206 form 3 additionalsalt bridges with D450 in the REC2 domain and K785 andE755 within the WED domain, respectively (Fig. 3D). Moreover,Y197 also forms a hydrogen bond with K785, and the side chain ofT201 forms a hydrogen bond with the phosphate group of A-20 inthe crRNA (Fig. 3E). The frequent electrostatic interactions inthis region indicate the potential high-affinity binding betweenAcrVA4 and LbCas12a-crRNA.On the other side of the valley, the A-B (residues 175 through

182) and E-F (residues 216 through 219) loops form the secondwing to cover the BH domain (Fig. 3B). At this interface, residueY160 in the A strand forms a hydrogen bond to R887 in the BHconnecting loop, which concomitantly contacts with the adjacentW178 of AcrVA4 by π-cation interaction. In addition, the sidechain of R217 interacts with both the main chain and side chainof F884 by hydrogen bond and π-cation interaction, respectively(Fig. 3F). Collectively, the 3 interacting subregions contact multiple

domains of LbCas12a, which would thus lock the conformations ofthese domains to hinder the structural transition required for tar-get dsDNA unwinding and hybridization with the crRNA.Previous study has shown that AcrVA4 could efficiently inhibit

the activity of LbCas12a but is ineffective to Acidaminococcus sp.Cas12a (AsCas12a), both of which have been utilized for genomeediting in cells (29). By comparing the structures of LbCas12aand AsCas12a, we found the WED domain of AsCas12a harborsan insertion of 2 helices, which directly collides with the centralhelix and CTD of AcrVA4 (SI Appendix, Fig. S6 A and B).Therefore, AsCas12a is resistant to the inhibition by AcrVA4.We also compared the structures of the LbCas12a-crRNA

complex before and after AcrVA4 binding to analyze the potentialconformational changes induced by the interaction. No majorchanges were observed in the structures except some local move-ments of the REC1 and REC2 domains (SI Appendix, Fig. S7A).The REC1 domain slightly rotates toward the PAM-interacting(PI) domain, and the REC2 domain moves outward to leave theWED domain. This rearrangement displaces the 450-helix by an∼8.5-Å distance to allow for enough space for accommodatingAcrVA4 (SI Appendix, Fig. S7B).

Ultrahigh-Affinity Binding Is Required for LbCas12a Inhibition byAcrVA4. To verify our structural analysis, we performed muta-genesis studies on the key interacting residues within AcrVA4 totest their effects on Cas12a binding and inhibition (Fig. 4 and SIAppendix, Fig. S8). Among the 3 subregions, the C-D loop pro-trusion and the wing formed by A-B and E-F loops contribute themajority of strong charged interactions (Fig. 3 C–F). Therefore,we conducted mutations on these 2 subregions which should becrucial for the inhibitory activity of AcrVA4.

Fig. 2. AcrVA4 binds to the LbCas12a-crRNA complex with 2 different stoichiometries. (A) Schematic diagram of the LbCas12a domain architecture. Each domainis represented by a unique color and the same color code is used throughout the manuscript. (B) Overall density map of LbCas12a-crRNA-AcrVA4 form A complexin which an AcrVA4 homodimer binds to a single copy of the LbCas12a-crRNA binary complex. The LbCas12amolecule is colored by domains and the crRNA repeatregion is colored in black. The AcrVA4 dimer is colored by chains with the main interacting subunit in orange and the other in gray. (C) Representative 2D classaverages of LbCas12a-crRNA-AcrVA4 form A and form B complexes. (D) Structure of LbCas12a-crRNA-AcrVA4 form B complex in which an AcrVA4 homodimerbinds to 2 copies of the LbCas12a-crRNA complex. One copy of LbCas12a-crRNA is colored by domains and the other is in white.

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As shown by surface plasmon resonance (SPR) assays, thewild-type (WT) AcrVA4 did not bind apo LbCas12a but showedan ultrahigh affinity to the LbCas12a-crRNA binary complexwhich did not dissociate after binding (KD < 0.09 pM) (Fig. 4 Aand B). This is consistent with the results of SEC assays thatAcrVA4 forms a stable complex with the crRNA-loaded butnot apo LbCas12a (Fig. 1 D and E). The affinity of AcrVA4 tocleavage-inactivated dLbCas12a-crRNA-dsDNA complex was∼0.12 pM, similar to that for the LbCas12a-crRNA binary com-plex (Fig. 4C). This observation suggested that AcrVA4 couldefficiently interact with the LbCas12a full R-loop complex andpossibly revert its conformation back to the crRNA-loaded stateby displacing the bound dsDNA. We also conducted LbCas12a-crRNA-mediated dsDNA cleavage in vitro and tested the in-hibitory effect of AcrVA4. The WT AcrVA4 inhibited the dsDNAcleavage in a dose-dependent manner, with an EC50 concentrationof ∼65.6 nM, against 100 nM LbCas12a-crRNA complex (Fig. 4I).At a concentration of 400 nM, ∼6 times the EC50 dose, it showedan almost complete inhibition for cleavage (Fig. 4J). Substitu-tions of K202 and K203 by alanine enabled the AcrVA4 mu-tant to dissociate after binding, and reduced the affinity by∼100,000 folds (Fig. 4D). Consistent with this observation, theinhibitory efficiency was obviously compromised with ∼50% and∼80% inhibitions at 800 and 1,600 nM (12 times and 24 times theEC50) concentrations, respectively (Fig. 4J). The E204A/R206Adouble mutant showed a 10-fold decrease in binding affinity andstill maintained good inhibitory efficiency for dsDNA cleavage(Fig. 4 E and J). Simultaneous substitution of the 4 residues re-duced the binding affinity by ∼1,000,000 folds in comparison tothe WT AcrVA4, which completely abolished the inhibition at 12-times the EC50 concentration and showed only weak inhibition at24 times the EC50 concentration (Fig. 4 F and J). On the otherhand, replacement of the 3 residues Y160, W178, and R217, which

form the right wing to interact with the BH domain, significantlyreduced the binding affinity by 1,000,000 folds compared to theWT and lost the inhibitory activity even at 24 times the EC50concentration (Fig. 4 G and J), comparable to the performanceof the quaternary mutant on the C-D loop. Replacing the 7 resi-dues with alanine simultaneously further reduced the binding af-finity to show a 10,000,000-fold decrease as compared to the WTAcrVA4 and completely lost the inhibitory effect at 24 times theEC50 concentration (Fig. 4 H and J). These data revealed thathigh-affinity binding is required for the efficient inhibition whichmay tightly lock the conformation of multiple LbCas12a domainsto disable its enzymatic activity.

Inhibition Mechanism of AcrVA4. Previous studies have shown thatcrRNA loading transforms the structure of Cas12a from extendedto compact conformation (36); the AcrVA4 binding valley locatedat the junction region between 2 Cas12a lobes (REC and NUClobes) thus could not be formed in the extended apo LbCas12a.The binding of the dsDNA target is coupled with further confor-mational changes in the Cas12a-crRNA binary complex to allowhybridization between the crRNA spacer and target strand (TS) ofdsDNA and to expose the nuclease active site between Nuc andRuvC nuclease domains (Fig. 5A) (32, 33, 35). In this transition,the conformation of the NUC lobe does not substantially changeexcept the BH and PI domains, whereas the domains within theREC lobe are significantly reoriented. The REC1 domain rotatestoward the PI domain and the REC2 domain flips upward to de-tach from the NUC lobe, thus creating a central channel to ac-commodate the crRNA-TS heteroduplex. In addition, the BH helixis elongated by the folding of connecting loop and moves towardthe REC lobe to make contacts with the REC2 domain andcrRNA-TS heteroduplex (Fig. 5A), which has been shown essentialfor dsDNA cleavage (35).

Fig. 3. Contacting interface between AcrVA4 and the LbCas12a-crRNA complex. (A) Atomic structure of the LbCas12a-crRNA-AcrVA4 form A complex. TheLbCas12a molecule is shown in surface model and colored by domains. The crRNA is represented by cartoons in which the CRISPR repeat and spacer regionsare colored in black and red, respectively. The bound AcrVA4 dimer is shown as ribbons and colored by chains of which the main contacting protomer iscolored in orange and the other in gray. (B) Close-up view of the binding interface. (C–F) Interaction details between AcrVA4 and the LbCas12a-crRNAcomplex. The key interacting residues are shown as sticks and colored by atoms. Hydrogen bonds and salt bridges are represented by black dashed lines.

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We then compared the structure of AcrVA4-bound LbCas12a-crRNA with the ternary complex of LbCas12a-crRNA-dsDNA bysuperimposing the NUC lobe. Despite that the conformation ofthe WED domain is well maintained after dsDNA binding, the450-helix in REC2 domain, the 895-helix in the RuvC domain, andthe BH helix are substantially rearranged, which are key structuralelements for AcrVA4 binding and inhibition (Fig. 5 B and C). Theconformation of the WED domain is preserved in the LbCas12a-crRNA-dsDNA ternary complex (SI Appendix, Fig. S6C), whichmight provide an initial binding site for AcrVA4 to release thebound DNA and possibly revert the conformation of LbCas12aback to the crRNA-loaded state (SI Appendix, Fig. S1B). In ad-dition, the deformation of the AcrVA4-contacting interface in theREC2, RuvC, and BH domains in the LbCas12a-crRNA-dsDNAcomplex indicates the ultrahigh-affinity binding of AcrVA4 to theLbCas12a-crRNA binary complex would indeed prevent confor-mational changes of these domains, which as a result would blockdsDNA binding (Fig. 5 B and C). This is supported by the ob-servation that unlocking the interactions by mutations in the C-Dloop protrusion and the A-B/E-F loop wing impaired the in-hibitory activity (Fig. 4J).

To further test the hypothesis, we performed electropho-retic mobility shift assays (EMSA) to test the binding betweenLbCas12a-crRNA-AcrVA4 and dsDNA. As the positive control,the dLbCas12a-crRNA complex efficiently interacted with dsDNAand showed a dose-dependent dsDNA retardation profile (Fig.5D). In contrast, the presence of AcrVA4 significantly impairedthe dsDNA binding activity of the dLbCas12a-crRNA complexand displayed very weak binding at high concentration of thecomplex (Fig. 5E). This result indicated that AcrVA4 bindinghinders the full R-loop formation but possibly allows PAM rec-ognition at the early stage of dsDNA binding, which does notrequire structural rearrangement in the AcrVA4-binding interfaceof LbCas12a.Collectively, these structural and biochemical evidences dem-

onstrate that AcrVA4 could disable LbCas12a-mediated CRISPR-Cas immunity at different stages of the working pathway (Fig. 6).First, AcrVA4 locks the conformation of crRNA-loaded LbCas12awith ultrahigh-affinity binding, preventing the dsDNA targetengagement and cleavage. Second, AcrVA4 can interact withthe LbCas12a-crRNA-dsDNA (full R-loop) complex to releasethe bound DNA before cleavage. In addition, it can also bind

Fig. 4. Mutagenesis verification of the interactions and effects on Cas12a inhibition. (A–C) SPR measurement for the binding affinity between AcrVA4 andLbCas12a at different states. (D–H) The binding kinetics of AcrVA4 mutants to LbCas12a-crRNA binary complex. The raw binding curves (black) and the fittedcurves (red) are superimposed in the figures. (I) Dose-dependent inhibition of AcrVA4 for LbCas12a-crRNA-mediated dsDNA cleavage in vitro. The LbCas12a-crRNA complex was used at 100 nM concentration in the reaction. The EC50 concentration of AcrVA4 was calculated based on the fitted curve. The error barsrepresent SDs of 3 independent experiments. (J) Effects of mutations on the inhibitory activities of AcrVA4 for LbCas12a-crRNA-mediated dsDNA cleavage.

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the postcleavage complex (cleaved R-loop) and probably block therecycling usage of the enzyme in the next round of catalysis.

DiscussionSo far, quite a few Acr proteins targeting different CRISPR-Cassystems have been identified and characterized, and the inhibitionmechanisms vary for different types of CRISPR-Cas systems (19).For example, AcrIF1 targets the type I-F surveillance complex toblock target DNA recognition by interfering with the base pairingbetween spacer and target (38, 39); AcrIF2 and AcrIF10 competewith target dsDNA for the binding site on the surveillance com-plex (38, 40); AcrIF3 binds to Cas3 endonuclease to prevent beingrecruited by the surveillance complex thereby blocking targetDNA access (41, 42); AcrIIA2 and AcrIIA4 prevent target DNArecognition of Cas9-guide RNA (gRNA) complex by dsDNAmimicking (43–45); AcrIIC1 inactivates the Cas9 activity byblocking the catalytic site (46); and AcrIIC2 interferes with theguide RNA loading process of Cas9 which consequently impairstarget DNA binding (47). AcrIIC3 dimerizes Cas9-gRNA com-plexes and inhibits target DNA binding (46, 47).In this work, we describe a comprehensive mechanistic in-

sight into a Cas12a-targeted Acr protein, AcrVA4, which inhibitsCRISPR-Cas immunity at multiple stages of the interferenceprocess. It not only interferes with target DNA binding, but alsoreleases the bound dsDNA to revert its fate before cleavage. Inaddition, it may block the recycling of the Cas12a enzyme afterdsDNA cleavage. Thus, AcrVA4 utilizes a unique multistage in-hibition mechanism for silencing CRISPR-Cas immunity, which

differs from other known Acr proteins with mainly only 1 stageinterference activity. The multistage interference of AcrVA4 en-sures high efficiency of CRISPR-Cas inhibition, which mightrepresent a more sophisticated strategy selected after the long-term evolution. Of note, most of the Acrs represent individuallynovel protein folds, suggesting the diverse origin of different Acrproteins. Based on sequence analysis, AcrVA4 could only befound in the genome ofMoraxella bovoculi and no other homologscould be detected. Interestingly, we identified a hypotheticalprotein in the same bacterium with ∼30% sequence identity,which potentially represents an AcrVA4 isoform or a novel Acrprotein (SI Appendix, Fig. S9).Among all these Acr proteins characterized so far, quite a

number of them intercept the recognition and binding of targetDNA, which thus represents an efficient and ubiquitous strategyto silence CRISPR-Cas immunity. However, this could beachieved through various mechanisms by different Acr proteins.AcrIF1 hinders the base pairing between the spacer and TS bysteric hindrance and inducing local conformational changes ofthe surveillance complex (38, 39). AcrIF2, AcrIF10, AcrIIA2,and AcrIIA4 mimic the structure of dsDNA to directly competefor the binding site of dsDNA target on the effector complexes(38, 40, 43–45). A recent work showed that AcrVA5 acetylatedlysine in the PI domain of Cas12a, thus conferring steric hin-drance for dsDNA binding (30). The observation that AcrVA4binds at the opposite side of the dsDNA binding site without directspatial competition is quite remarkable, which adopts a noncom-petitive mechanism to inhibit target DNA binding by locking the

Fig. 5. AcrVA4 binding locks the conformation of the LbCas12a-crRNA binary complex and interferes with dsDNA binding. (A) The conformational changesof LbCas12a-crRNA before (Left, in dark color) and after (Right, in light color) dsDNA binding. The conformation of the NUC lobe does not significantly change(shown as transparent surface), whereas the REC lobe is substantially reorganized (in cartoon model, colored by domains). The key motifs involved in thetransition are shown as cartoons. The directions for structural transition are indicated by arrows with the same colors of corresponding domains. The spacer ofcrRNA is colored in red, and the TS and NTS of dsDNA are colored in purple and cyan, respectively. The catalytic site is highlighted with a red asterisk. (B)AcrVA4 (orange, transparent) binds to the LbCas12a-crRNA binary complex and interacts with the BH domain, 895-helix, and the REC2 domain to preventtheir structural rearrangement for dsDNA binding. (C) Superposition of the LbCas12a-crRNA-AcrVA4 with LbCas12a-crRNA-dsDNA complex. The close-up viewat the AcrVA4-interacting interface is shown to reveal the conformational changes of the BH domain, the 895-helix of RuvC domain, and the 450-helix of theREC2 domain, which are locked by AcrVA4 binding. The key interacting residues in LbCas12a are shown in sticks to highlight the deformation of theAcrVA4 binding site after dsDNA binding. (D and E) EMSA assay to test the binding of dsDNA to the dLbCas12a-crRNA complex in the absence (D) or presence(E) of AcrVA4. The NTS was labeled with Cy5 fluorophore at the 5′-end. Protein complexes were used with gradient concentrations.

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conformation of the CRISPR effector complex. During thereviewing process of our manuscript, Zhang et al. (48) reportedsimilar results that AcrVA4 prevents the conformational changesof Cas12a for R-loop formation. This unique inhibition mecha-nism is achieved by the ultrahigh binding affinity that resultsfrom multiple contacting interfaces and the occurrence of fre-quent salt bridge interactions.The binding affinity of AcrVA4 to Cas12a is extraordinarily

high among all of the Acr proteins characterized so far, eventhough the affinities of these Acrs are universally as high asnanomolar range (38, 45–49). As the SPR assays revealed non-dissociative binding kinetics for AcrVA4, the affinity value couldnot be precisely calculated but indeed was beyond nanomolar scale.We further verified the binding affinities by biolayer interferometry(BLI) analysis and similar nondissociative kinetics were observed(SI Appendix, Fig. S10), suggesting the interactions betweenAcrVA4 and Cas12a might be irreversible at any one stage of theworking pathway. This highlights the fact that the conformationallocking mechanism requires ultrahigh binding affinity of the in-hibitor, as binding itself is not sufficient for the inhibition. Theseunique noncompetitive features offer the guidelines for designingdedicate “off-switch” tools for Cas12a-based genome-editing andDNA-targeting platforms. In addition, it could be utilized forprogramming specific phages to treat challenging infections ofbacteria with CRISPR-Cas12a systems.It is noteworthy that most Acr proteins characterized so far

directly interact with CRISPR effector complexes. However,there are other checkpoints in the CRISPR-Cas working path-way that could be targeted to block the immunity. It has beenshown that AcrIIA1 interacts with RNA but the extract workingmechanism is unclear (50). Possibly it affects the crRNA loadinginto the Cas9 effector and inhibits the assembly of the func-tional CRISPR-Cas effector complex. Besides, suppression ofpre-crRNA transcription/processing and Cas proteins expres-sion/recycling, or target nucleic acid shielding, could also befeasible strategies to achieve CRISPR-Cas inhibition. Further

work in the field would greatly expand our understanding of thefunctionality of these systems and stimulate new applications.

Materials and MethodsMore detailed descriptions of the materials and methods used in this studyare provided in SI Appendix. A brief summary is provided here.

Protein Expression and Purification. The coding sequences for AcrVA4 (NCBIEntry: WP_046699156) and LbCas12a (NCBI Entry: WP_035635841) weresynthesized and codon optimized for expression in E. coli. AcrVA4 wasexpressed with an N-terminal 6×His tag, and LbCas12a was fused with amaltose binding protein (MBP) tag, an N-terminal 6×His, and C-terminal2×Strep tag to facilitate purification. The crRNA was produced with an ad-ditional plasmid which was cotransformed with Cas12a to enable self-processing into mature crRNA. All proteins were solubly expressed inE. coli BL21 (DE3) at 16 °C and purified by tandem affinity chromatography,ion-exchange chromatography, and size-exclusion chromatography (SI Ap-pendix, SI Materials and Methods). All protein samples were analyzed bySDS-PAGE and reached a purity of ∼95%.

Cryo-EM Data Collection, Image Processing, and Model Building. The LbCas12a-crRNA-AcrVA4 complex was prepared and vitrified using a FEI Vitrobot MarkIV. Cryo-EM datawere collected with a 300 kV Titan Krios transmission electronmicroscope equippedwith a GIF-Quantumenergy filter and aGatan K2-summitdirect electron detector (SI Appendix, SI Materials and Methods). Images ofprocessing and reconstruction were performed using RELION-3.0 (51). Modelbuilding and refinement were conducted with CHIMERA (52), COOT (53), andPHENIX (54) (SI Appendix, SI Materials and Methods). Structural figures wererendered by PyMOL (https://pymol.org/) or CHIMERA (52). Representativedensities are shown in SI Appendix, Fig. S4. The statistics for image processingand model refinement are summarized in SI Appendix, Table S1.

SPR Assay. SPR experiments were performed at room temperature (r.t.) usinga Biacore 8K system with SA chips (GE Healthcare). LbCas12a at differentstates (apo LbCas12a, LbCas12a-crRNA complex, dLbCas12a-crRNA-dsDNAcomplex, and LbCas12a-crRNA-dsDNA complex) was immobilized on the chipby biotin affinity tag. The single-cycle binding kinetics for WT AcrVA4 andmutants was analyzed using a 1:1 (a copy of LbCas12a-crRNA versus anAcrVA4 dimer) binding model (SI Appendix, SI Materials and Methods).

Fig. 6. Schematic model of AcrVA4 inhibiting Cas12a-mediated dsDNA cleavage. The models of Cas12a are colored by domains as annotated and theAcrVA4 dimer is shown with 2 protomers in different colors. AcrVA4 could inhibit the activity of Cas12a at several steps in the catalytic cycle. It binds theLbCas12a-crRNA binary complex to prevent dsDNA engagement. Despite that PAM recognition is not prohibited, further dsDNA unwinding and base pairingwith the crRNA spacer are perturbed to block full R-loop formation. Moreover, it could also interact with the LbCas12a-crRNA-dsDNA ternary complex (full R-loop state) to release the bound dsDNA before cleavage. This process could possibly revert the conformation of LbCas12a back to the pre-dsDNA bindingstate. In addition, AcrVA4 also binds to the cleaved R-loop complex to block the recycling of the enzyme. Thus, AcrVA4 utilizes different mechanisms forinhibiting LbCas12a activity at multiple stages of CRISPR-Cas immunity.

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In Vitro Cleavage Assay. The in vitro dsDNA cleavage assayswere conducted in acleavage buffer consisting of 20 mMHepes-NaOH, pH 7.5, 150 mMKCl, 10 mMMgCl2, and 0.5 mM Tris(2-carboxyethyl)phosphine (TCEP). The linearizedplasmid and serially diluted AcrVA4 protein were preincubated at 37 °C for10 min, and the LbCas12a-crRNA binary complex was then added to start thereaction. After an additional 30-min incubation at 37 °C, the reaction systemwas resolved by electrophoresis using a 1% agarose gel and visualized bystaining with SYBR Green dyes (Invitrogen) (SI Appendix, SI Materials andMethods). The images were quantified by integrating the intensity of eachband using ImageJ software. The dose-dependent inhibition curve was gen-erated with GraphPad Prism 5 to estimate the EC50 value.

Data Availability. The cryo-EM density maps of form A and form B LbCas12a-crRNA-AcrVA4 complexes have been deposited to the Electron MicroscopyData Bank (EMDB) with the accession codes EMD-0704 and EMD-0705,respectively. The coordinates of corresponding atomic models have beendeposited to the Protein Data Bank (PDB) under the entries 6KL9 and6KLB, respectively.

ACKNOWLEDGMENTS. We thank all staff at the Center of Biological Imaging,Institute of Biophysics (IBP), Chinese Academy of Sciences (CAS), Beijing, andthe cryo-EM facility of Southern University of Science and Technology,Shenzhen, for assistance with data collection. We are grateful to Dr. GuopengWang (The Core Facilities at School of Life Sciences, Peking University) and Dr.Tie Yang and staff in the EM department of the State Key Laboratory ofMembrane Biology, Institute of Zoology, CAS, Beijing, for technical support inthe electron microscope operation. We appreciate the help of Xiaomin Wangand Manling Zhang in preparing reagents, as well as Yuanyuan Chen andZhenwei Yang at the Core Facility for Protein Research (IBP, CAS) for assistancewith Biacore experiments. This study was supported by the Strategic PriorityResearch Program of CAS (XDB29010000), the National Science and Technol-ogy Major Project (2018ZX10101004), and the Young Scientist Fund Project ofthe National Natural Science Foundation of China (NSFC, 81802010). R.P. issupported by the Young Elite Scientist Sponsorship (YESS) Program by theChina Association for Science and Technology (CAST) (2018QNRC001). Y.S. issupported by the Excellent Young Scientist Program from the NSFC(81622031), the Excellent Young Scientist Program, and the Youth InnovationPromotion Association of CAS (2015078). G.F.G. is supported partly as a leadingprincipal investigator of the NSFC Innovative Research Group (81621091).

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