Structure Studies of the CRISPR-Csm Complex Reveal Mechanism of Co-transcriptional Interference

Abstract

Csm, a type III-A CRISPR-Cas interference complex, is a CRISPR RNA (crRNA)-guided RNase that also possesses target RNA-dependent DNase and cyclic oligoadenylate (cOA) synthetase activities. However, the structural features allowing target RNA-binding-dependent activation of DNA cleavage and cOA generation remain unknown. Here, we report the structure of Csm in complex with crRNA together with structures of cognate or non-cognate target RNA bound Csm complexes. We show that depending on complementarity with the 5' tag of crRNA, the 3' anti-tag region of target RNA binds at two distinct sites of the Csm complex. Importantly, the interaction between the non-complementary anti-tag region of cognate target RNA and Csm1 induces a conformational change at the Csm1 subunit that allosterically activates DNA cleavage and cOA generation. Together, our structural studies provide crucial insights into the mechanistic processes required for crRNA-meditated sequence-specific RNA cleavage, RNA target-dependent non-specific DNA cleavage, and cOA generation.

Keywords: CRISPR-Cas 9 system; Csm complex; type III-A.

Figure 1.. Crystal Structure of the SthCsm Complex

(A) Schematic view of a type III-A CRISPR-Cas system in S. thermophilus. (B) The overall structure of the SthCsm complex associated with a crRNA. Color coding used for Csm1–Csm5 is identical to that used in (A). The spacer and 5′ tag regions of crRNA are in red and gray, respectively. (C) The schematic representation of Csm1 domains. The HD, Palm1, Linker, Palm2, and D4 domains are shown in cyan, orange, purple, salmon, and wheat, respectively. (D) Structure of Csm1 subunit in the same orientation as in (B). (E) Close-up view of the crRNA spacer region and Csm3 β-thumb. The β-thumb of each Csm3 subunit folds over the top of the crRNA, creating a kink in the crRNA at 6-nt interval. (F) Magnified view of the interactions between nucleotides (−6)–(−8) and the Csm1-Csm4 subcomplex illustrates that nucleotides (−6)–(−7) are sequence specifically recognized by Csm1 and Csm4 subunits. (G) The target RNA dependent non-specific DNA cleavage with either Csm4 or Csm1 mutant form. (H) Interactions between the nucleotides (−2)–(−5) of crRNA and Csm4-Csm3.1 subcomplex showing that these nucleotides are solvent exposed. See also Figure S1 and Table S1.

Figure 2.. Cryo-EM Structure of the Csm-NTR Complex

(A) Schematic representation of the crRNA-NTR duplex. The base pairs observed in the NTR-bound Csm complex are depicted by lines. The non-cognate target RNA is shown in blue. The ordered and disordered nucleotides of NTR are shown in white and black, respectively. (B) Overall structure of the NTR-bound Csm complex. (C) The β-thumb of each Csm3 subunit inserts into the crRNA-NTR duplex, leading to the periodical nucleotide displacement of both RNA strands. (D) Structural comparison between Csm-crRNA binary complex and Csm-crRNA-NTR ternary complex showing the conformational change upon target RNA binding. Vector length correlates with the domain motion scale (color-coded as defined in Figure 1A). (E) Nucleotides at positions (−2)′–(−5)′ in the 3′ anti-tag of NTR base pair with nucleotides (−2)–(−5) in the 5′ tag of the crRNA. (F) The side-chain of Arg253 of Csm4 subunit inserts into the bases at position (−6) of crRNA and target RNA, preventing base pairing at this position. See also Figures S2 and S3 and Table S2.

Figure 3.. Cryo-EM Structure of the Csm-CTR Complex Either in the Absence or Presence of DNA

(A) Schematic representation of the crRNA-CTR1 duplex. The color codes of crRNA and target RNA are identical to those used in Figure 2A. (B) Overall structure of Csm1 H15A and Csm3 D33N catalytic mutant Csm in complex with CTR2 in the presence of bubble dsDNA at 3.05 Å resolution. The disordered the 3′ anti-tag of CTR2 is shown by a blue dashed line. (C) Cryo-EM maps and fits for selected regions of Csm1 (residues 719–734) and Csm2.1 (residues 106–122) in the 3.05 Å CTR-bound Csm complex. (D) Schematic representation of the Csm-CTR complex. Color-coding is identical to that used in Figure 1B. (E) Close-up view of the scissile phosphate and the conserved catalytic residue Csm3 Asp33. The Csm3 D33N mutant was used for structural studies. (F) Target RNA cleavage assay. Three scissors indicate the cleavage sites. The Csm1 mutants are in black, and Csm2 and Csm3 mutants are highlighted in yellow and cyan, respectively. See also Figure S4 and Table S2.

Figure 4.. Cryo-EM Structure of the Csm-CTR Complex in the Presence of ATP or AMPPNP

(A) Overall structure of Csm1 D16N and Csm3 D33N catalytic mutant Csm in complex with cognate target RNA in the presence of AMPPNP. (B) The non-complementary 3′ anti-tag region of the CTR binds in a positively charged groove of the Csm1 subunit, which is shown as a surface representation colored according to its electrostatic potential. (C) Close-up view of the interaction between the 3′ anti-tag region of the CTR and Csm1 subunit. (D) Nonspecific ssDNA cleavage with Csm1 mutants showing the impact of mutation in the Csm1 Linker region and Loop L1 on HD nuclease activity. WT, wild-type Csm; NTR, non-cognate target RNA bound wild-type Csm complex; ΔZF, Ser substitution of four Cys residues in the zinc finger. WT and mutant forms are CTR-bound Csm complexes containing either wild-type or mutant Csm1. (E) Effect of mutations in Csm1 on the cOA synthesis. Averages with SDs are shown, n = 3 replicates. (F) The DNA cleavage assay showing ATP has little impact on the DNA cleavage with either the wild-type (left) or mutant Csm complex (right). (G) RNA cleavage assay showing the Csm1 mutation has little impact of on target RNA cleavage. (H) DNA cleavage assay showing the impact of 3′ anti-tag mutation on the DNase activity of Csm1 HD domain. (I) Effect of mutations of the 3′ anti-tag sequence in CTR on cOAs synthesis. Averages with SDs are shown, n = 3 replicates. See also Figure S5 and Table S3.

Figure 5.. Two ATP or AMPPNP Bind in the Palm Domains of Csm1 Subunit

(A) Two AMPPNP molecules bind in the pocket formed by two Palm domains. AMPPNP1 (in green) and AMPPNP2 (in teal) bind to Palm1 and Palm2 domains, respectively. The a-phosphate group of AMPPNP2 and the O3′ atom of AMPPNP1 are shown in yellow and red, respectively. (B) Cryo-EM map for two AMPPNP molecules in the CTR-bound Csm complex (left) and for two ATP in the NTR-bound Csm complex (right). Two Mg²⁺ are shown as brown spheres. (C) Csm4 residues 82–104 (in magenta) cover the ATP binding pocket upon ATP binding, whereas they are disordered in the absence of ATP. (D) Structural comparison the GGDD motif in the CTR-bound Csm complex either in the presence (wheat) or absence (gray) of AMPPNP, showing that the GGDD motif undergoes a conformational change upon the AMPPNP or ATP binding. (E) Magnified view of the interactions between AMPPNP1 and the Palm1 domain. (F) Expanded view of the interactions between AMPPNP2 and the Palm2 domain. (G) Effect on cOAs synthesis of mutations of Csm1 residues that interact with the AMPPNP1. (H) Effect on cOAs synthesis of the mutations of Csm1 and Csm4 residues that interact with the AMPPNP2. The Csm4 mutants are highlighted in green. See also Figure S5.

Figure 6.. Determining the Minimal Length of crRNA Spacer Segment Required for DNase and cOA Synthetase Activities of the Csm Complex

(A and B) DNA cleavage by various CTR-bound SthCsm complexes. The cognate target RNA is truncated from it 5′ end (A), and the target RNA contains mismatches from its 5′ end (B). (C) cOAs synthesis assay with truncated cognate target RNA shown in (A). (D) cOAs synthesis assay with the mismatch-containing cognate target RNA as shown in (B). (E) Efficiency of transformation (EOT) of III-A⁺ T. thermophilus cells with plasmids carrying protospacers fully matched to (“WT”) or with multiple mismatches with the 3′ end of crRNA. Numbers indicate the length of crRNA-target duplex. Blue bars correspond to plasmids with protospacer cloned in direct orientation, which results in high level of transcripts targeted by crRNA, red bars correspond to plasmids with reverse protospacer orientation which results in reduced protospacer transcript levels. The gray bar indicates EOT with control plasmid without protospacer (“No PS”). The structures of crRNA-target duplexes for fully matching protospacers in both orientations are shown on the right. Mean values obtained from 3 independent experiments and SDs are shown. See also Figure S6.

Figure 7.. Non-complementarity of 3′ Anti-tag of Target RNA Activates the DNase and cOA Synthetase Activities

(A) Structural comparison between NTR- and CTR-bound Csm complexes. Superposition of the NTR- and CTR-bound Csm complexes at the repeat region. The 5′ tag and 3′ anti-tag in the CTR-bound Csm complex are shown in red and blue, respectively. The Color-coding of Csm1 in the CTR-bound complex is identical to that used in Figure 1C. All components in the NTR-bound complex are shown in gray. Two green arrows highlight the clash region of between NTR and Csm1 within CTR-bound complex. (B) Structural comparison of HD domains in the CTR- (cyan and purple) and NTR-bound (gray) Csm complexes by aligning the palm2 and D4 domains of the Csm1 subunit. (C) Structural comparison of Palm domains in the CTR- (orange and salmon) and NTR- (gray) bound Csm complexes by aligning the palm2 and D4 domains of the Csm1 subunit. (D) Model of type III-A interference. The crRNA associated with the Csm complex recognizes target RNA containing its complementary sequence forming the crRNA-target RNA duplex. The Csm3 subunit is an RNase that cleaves target RNA at 6-nt intervals. The 3′ anti-tag region of cognate target RNA binds to the Csm1 Linker and Loop L1 regions (shown in magenta triangles) and induces the rearrangement at the Linker and Loop L1 regions. This rearrangement of Csm1 allosterically activates the DNA cleavage and the production of cOAs, which activates the RNase activity of Csm6. See also Figure S7.