CRISPR-Cas and cGAS-STING Surveillance Pathways

CRISPR-Cas and cGAS-STING Surveillance Pathways


This section addresses our contributions to a mechanistic understanding of the CRISPR-Cas and cGAS-STING surveillance pathways.

CRISPR-Cas Surveillance Complexes

Efficient and site-specific genome engineering can be achieved based on programmable nucleic acid cleavage using CRISPR-Cas surveillance complexes. Structure-function studies on single component Cas (types V and VI) and multi-component Cascade (types I and III) are shedding light on the principles underlying cleavage chemistry of dsDNA and ssRNA targets. Ongoing challenges include a molecular understanding of the diverse mechanisms adopted by distinct CRISPR-Cas systems in efforts to broaden and enhance their applicability as genome editing tools. These efforts are being extended to CRISPR-transposition systems and to provide an understanding of recognition principles involving evolved viral suppressor proteins that inhibit the CRISPR-Cas pathway. We have also investigated the role of accessory nucleases in cleaving ssRNA and ssDNA and the principles underlying their regulation.


Type V Cas12 and Type VI Cas13 Systems. The earlier research on the dsDNA cleavage activity of class 2 type II Cas9 endoribonucleases has been recently extended to class 2 type V Cas12a (Cpf1) and Cas12b (C2c1) counterparts. The Cas12 enzymes are distinctly different from their Cas9 counterparts since (1) Cas12 enzymes contain only one RuvC domain but lack the HNH domain observed in Cas9; (2) Cas12 enzymes recognize distal 5’-T-rich PAMs, in contrast to a proximal 3′-G-rich PAM recognition by Cas9; and (3) Cas12 enzymes make staggered double-strand breaks, whereas Cas9 generates blunt ends. We have undertaken structural studies on ternary complexes of Cas12a (Gao et al. 2016) and Cas12b (Yang et al. 2016) with guide RNA and target dsDNA in efforts to provide a molecular basis for efforts towards harnessing these endoribonucleases for genome editing in mammalian cells. In particular, we have identified base-specific recognition of the PAM sequence and the positioning of target and non-target DNA strands in the RuvC catalytic pocket (Yang et al. 2016).

Type VI Cas13 is atypical in that it uses guide and target RNAs to cleave substrate RNAs within a composite HEPN pocket. Our structural studies have highlighted the role of tag:anti-tag pairing complementarity in dictating cleavage of viral RNAs, but cleavage suppression of host RNAs (Wang et al. 2021).

Gao, P., Yang, H., Rajanshankar, K. R., Huang, Z. & Patel, D. J. (2016). Type V CRISPR-Cas Cpf1 endonuclease employs a unique mechanism for crRNA-mediated target recognition. Cell Research 26, 901-913.

Yang, H., Gao, P., Rajashankar, K. R., & Patel, D. J. (2016). PAM-dependent target DNA recognition and cleavage by C2c1 CRISPR-Cas endonuclease. Cell 167,1814-1828.

Wang, B., Zhang, T., Yu, Y., Xu, W., Ding, J. Patel, D. J. & Yang, H. (2021). Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in Type VI Cas13 CRISPR systems. Mol. Cell 81, 1100-1115.


Type I Csy and Type III Csm Cascade Systems. Our group has undertaken cryo-EM studies of type I-F crRNA-bound Csy Cascade complex in the absence and presence of dsDNA in collaboration with the Sriram Subramaniam lab (NCI) (Guo et al. 2017). We observe ternary and quarternary conformational transitions on complex formation with dsDNA that may contribute to target dsDNA degradation by a trans-acting helicase-nuclease.

Our cryo-EM studies on crRNA-bound type III-A Csm Cascade complexes in the absence and presence of target RNA have demonstrated that the topological features of the 5’-repeat tag of crRNA provides insights into complex assembly and autoimmunity (Jia et al. 2019a). We also elucidate mechanisms underlying Csm3 subunit-mediated target cleavage with 6-nucleotide periodicity and the contribution of a Glu-rich loop within the Csm1 subunit HD pocket to regulation of DNase cleavage activity. We have also investigated formation of second messenger cA4 within the Csm1 PALM pocket and identified it release path (Jia et al. 2019b).

Guo, T. W. et al., Patel, D. J. & Subramaniam, S. (2017). Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Cell 171, 414-426.

Jia, N., Wang, C., Mo, C. Y., Eng, E. T., Marraffini, L. A. & Patel, D. J. (2019a). Type III-A CRISPR Csm complexes: Assembly, target RNA recognition, periodic cleavage and autoimmunity. Mol. Cell 73, 264-267.

Jia, N., Jones, R., Sukenick, G. & Patel, D. J. (2019b). Second messenger cA4 formation within the composite Csm1 Palm pocket of type III-A CRISPR-Cas Csm complex and its release path. Mol. Cell 75, 933-943.


CRISPR-Transposition Systems. Recent bioinformatics analyses have revealed the presence of CRISPR-Cas loci in bacterial Tn7-like transposons, thereby implicating a functional relationship between RNA-guided DNA targeting and transposition, with the latter representing a new role unrelated to host defense. Our cryo-EM studies of transposon subunit TniQ bound to crRNA- and dsDNA-bound type I-F Cascade established binding of a TniQ dimer to one end of the Cascade through contacts with the Cas7.1 and Cas6 subunits, thereby providing insights into the initial step of DNA integration by a CRISPR-Cas-transposon complex (Jia et al. 2020).

Jia, N., Xie, W., de la Cruz, M. J., Eng, E. T. & Patel, D. J. (2020). Structure-function insights into the initial step of DNA integration by a CRISPR-Cas-Transposon complex.  Cell Research 30, 182-184.


Accessory CRISPR Nucleases. Type III-A CRISPR-Cas surveillance complexes containing multi-subunit Csm effector protein together with guide and target RNAs exhibit multiple activities, including cAn-mediated cleavage of ssRNAs by trans-acting Csm6 RNase. Our crystallographic studies of dimeric T. onnurineus Csm6 in the apo- and cA4-bound states has provided mechanistic insights into how cA4 binding to the CARF domain triggers the RNase activity of the HEPN domain (Jia et al. 2019c). We further demonstrate that the Csm6 CARF domain is a ring nuclease, whereby bound cA4 positioned in the CARF domain is cleaved to ApA>p, with such cleavage bursts using an unprecedented timer mechanism to regulate the cleavage activity of the HEPN domain.

We also show that dimeric Card1 which contains CARF and REase domains on activation by cA4 cleaves both ssRNA and ssDNA. Binding of cA4 to the CARF domain results in a conformational change comprising the rotation of individual monomers relative to each other to form a more compact dimeric scaffold (Rostol et al. 2021). Our collaborator Luciano Marraffini (Rockefeller University) established that activation of Card1 induces dormancy of the infected hosts to provide immunity against phage infection and plasmids.

Jia, N., Jones, R., Yang, G., Ouerfelli, O. & Patel, D. J. (2019c). CRISPR-Cas III-A Csm6 CARF domain is a ring nuclease triggering stepwise cA4 cleavage with ApA>p formation terminating RNase activity. Mol. Cell 75, 944-956.

Rostol, J.T., Xie, W., Kuryavyi, V., Kao, K., Fromm, R., Patel, D. J. & Marraffini, L. A. (2021). The Card1 nuclease provides bacterial defense during Type III CRISPR immunity. Nature 590, 624-629.


Anti-CRISPR Proteins. Bacteriophages have evolved a broad spectrum of anti-CRISPR proteins to counteract and overcome the CRIAPR-Cas surveillance pathway.

We have written a review on anti-CRISPR proteins and their functional implications for genome editing (Jia and Patel, 2021).

Jia, N. & Patel, D. J. (2021). Structure-based functional mechanisms and biotechnology applications of anti-CRISPR proteins. Nat. Rev. Mol. Cell Biol. 22, 563-579.

Our group has determined the crystal structure of the complex formed by anti-CRISPR protein AcrIIA4 and sgRNA-bound type II Cas9. We show that AcrIIA4 preferentially targets critical residues essential for PAM duplex recognition, as well as blocks access to key catalytic residues lining the RuvC pocket, thereby inactivating Cas9 (Yang & Patel, 2017).

We have also determined the cryo-EM structure of the complex formed by anti-CRISPR protein AcrVIA1 and crRNA-bound type VI Cas13a. Our studies establish that AcrVIA1 interacts with the guide exposed face of Cas13a, preventing access to the target RNA and conformational changes required for nuclease activation (Meeske et al. 2020). Our collaborator Luciano Marraffini of showed that a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.

We have investigated the binding of anti-CRISPR proteins to the crRNA-bound type I-F Csy Cascade system in collaboration with the Sriram Subramaniam lab. Our cryo-EM studies establish that AcrF10 binds to the DNA PAM recognition site on Cascade thereby preventing the first step of dsDNA recognition, while AcrF1 binds to two sites on the crRNA thereby occluding crRNA-target DNA pairing and R-loop formation (Guo et al, 2017).

Yang, H. & Patel, D. J. (2017). Inhibition mechanism of an anti-CRISPR suppressor targeting SpyCas9. Mol. Cell 67, 117-127.

Guo, T. W. et al., Patel, D. J. & Subramaniam, S. (2017). Cryo-EM structures reveal mechanism and inhibition of DNA targeting by a CRISPR-Cas surveillance complex. Cell 171, 414-426.

Meeske, A. J., Jia, N., Cassel, A., Kozlova, A., Liao, J., Wiedman, M., Patel, D. J. & Marraffini, L. A. (2020). Phage-encoded anti-CRISPR enables full escape from type VIA CRISPR-Cas immunity. Science 369, 54-59.


cGAS-STING Surveillance Complex

There is currently considerable interest towards gaining an improved molecular and functional understanding of innate immune sensors of higher metazoans that recognize nucleic acids in the cytoplasm and trigger type I interferon induction. Cytoplasmic dsDNA of pathogenic bacterial or viral origin, and perhaps also displaced nuclear or mitochondrial DNA following cellular stress, represent such a trigger. cyclic GMP-AMP synthase (cGAS) was identified by the Zhijian Chen lab (University of Texas Southwestern Medical School) as a cytoplasmic DNA sensor that activates the type I interferon pathway by synthesizing the second messenger cGAMP shown by our group to contain mixed 3’,5’ and 2’,5’ linkages. cGAS was shown to be a member of the nucleotidyltransferase family and to be capable of generating cGAMP in vitro from GTP and ATP in the presence of dsDNA. cGAMP in turn bound to and activated the adaptor STING, resulting in the activation of transcription factor IRF3 and subsequent induction of interferon beta. Our ongoing studies have focused on identifying and characterizing inhibitors of cGAS and STING as therapeutic agents.


cGAMP contains a 2’-5’ linkage. cGAMP was shown by the Zhijian Chen lab to be a metazoan second messenger triggering an interferon response. cGAMP is generated from GTP and ATP by cytoplasmic dsDNA sensor cGAS. Our group combined structural, chemical (lab of collaborator Roger Jones), biochemical (lab of collaborator Thomas Tuschl) and cellular (labs of collaborators Winfried Barchet and Gunter Hartmann) assays to demonstrate that this second messenger contains G(2’,5’)pA and A(3’,5’)pG phosphodiester linkages, designated c[G(2’,5’)pA(3’,5’)p] (Gao et al. 2013a). Our structural studies demonstrate that upon dsDNA binding, cGAS is activated through conformational transitions, resulting in formation of a catalytically competent and accessible nucleotide binding pocket for generation of cGAMP. We demonstrate that cyclization occurs in a stepwise manner through initial generation of 5’-pppG(2’,5’)pA prior to cyclization to c[G(2’,5’)pA(3’,5’)p], with the latter positioned precisely in the catalytic pocket. Mutants of cGAS dsDNA-binding or catalytic pocket residues exhibit reduced or abrogated activity.

Gao, P. et al., Jones, R. A., Hartmann, G., Tuschl, T. & Patel, D. J. (2013a). Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094-1107.


STING Activation by cGAMP. cGAMP has been shown by the Zhijian Chen lab to bind the signaling protein STING with subsequent activation of the IFN pathway. Our structural studies established that human hSTING undergoes a conformational change upon binding to cGAMP leading to formation of a ‘closed’ conformation capped by a b-sheet lid (Gao et al. 2013b). Comparing hSTING to mSTING, 2’,5’-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to their all-3’,5’-linkage counterpart. Guided by structural information, point mutations placed within the cGAMP binding pocket (S162A and Q266I) and the b-sheet lid (G230I) of hSTING, rendered it sensitive to the otherwise mouse-specific drug DMXAA. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.

Gao, P. et al., Hartmann, G., Barchet, W., Tuschl, T. & Patel, D. J. (2013) Structure-function analysis of STING activation by c[G(2’,5’)pA(3’,5’)p] and targeting by DMXAA. Cell 154, 748-762.


Additional DNA-binding Interface on cGAS. Studies by the Zhijian Chen lab have established that the positively charged N-terminal segment of cGAS contributes to enhancement of cGAS enzymatic activity as a result of DNA-induced liquid-phase condensation. We have identified an additional cGAS CD-DNA interface (labeled site-C; CD, catalytic domain), with mutations along this basic site-C cGAS interface disrupting liquid-phase condensation (Xie et al. 2019). We propose a multivalent interaction-mediated cluster model to account for DNA-mediated condensation involving both the N-terminal domain of cGAS and the site-C cGASDNA interface. We also report the crystal structure of human cGAS CD-DNA complex containing a triple mutant that disrupts the site-C interface, with this complex serving as a future platform for guiding cGAS inhibitor development.

Xie, W., Lama, L., Adura, C., Glickman, J. F., Tuschl, T. & Patel, D. J. (2019). Human cGAS catalytic domain has an additional DNA-binding interface that enhances enzymatic activity and liquid phase condensation. Proc. Natl. Acad. Scis. USA 116,11046-11955.


Inhibitors Targeting cGAS Scaffold. In a collaborative effort with the Thomas Tuschl lab (Rockefeller) we have identified human-cGAS-specific small-molecule inhibitors by high-throughput screening followed by targeted medicinal chemistry optimization (Vincent et al. 2017; Lama et al. 2019). Structural characterization has allowed us to position these small molecules in the cGAMP binding pocket of cGAS. These novel cGAS inhibitors with cell-based activity will serve as probes into cGAS-dependent innate immune pathways and warrant future pharmacological studies for treatment of cGAS-dependent inflammatory diseases.

Lama, L. et al., Glickman, J. F., Patel, D. J. & Tuschl, T. (2019). Development of hum DNA-mediated condensation an cGAS-specific small molecule inhibitors with biochemical and cell-based activity for repression of dsDNA-triggered interferon expression. Nat. Commun. 10: 2261.

Vincent, J. et al., Tuschl, T., Patel, D. J., Glickman, J. F. & Ascano, M. (2017). Small molecule inhibition of cGAS reduces interferon expression in primary macrophages from autoimmune mice. Nat. Commun. 8:750.


RIG-I Surveillance Complex

RIG-I is a cytosolic helicase that senses 5’-ppp-RNA contained in negative strand RNA viruses and triggers innate antiviral immune responses.


5’-ppp-RNA Recognition by RIG-I.

We have solved the crystal structure of RIG-I CTD domain bound to both blunt-ends of a self-complementary 5’-ppp-dsRNA 12-mer, with interactions involving 5’-pp clearly visible in the complex (Wang et al. 2010). The structure, supported by mutation studies undertaken in the lab of our collaborator Thomas Tuschl (Rockefeller University) defines how a lysine-rich basic cleft within the RIG-I CTD domain sequesters the observable 5’-pp of the bound RNA, with a stacked Phe capping the terminal base pair.

Wang, Y. et al., Micura, R., Tuschl, T., Hartmann, G. & Patel, D. J. (2010). Structural and functional insights into 5’-ppp-RNA pattern recognition by the innate immune receptor RIG-I. Nat. Struct. Mol. Biol. 17, 781-787.