Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9

Abstract

Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements.

Fig.1.. Cryo-EM reconstruction and structure of IscB RNP bound to target DNA.

(A) Arrangement of the OgeuIscB and ωRNA in its native IS element defined by the left (LE) and right (RE) ends of the transposon. (B) Domain organization of IscB. P1D, P1 interaction domain; TID, TAM-interaction domain. RuvC domain is separated into three segments: RuvC I, II, and III. Color scheme is conserved throughout Fig. 1. (C) Diagram of R-loop formed between guide RNA and target DNA. TAM sequence is read 5’-CTAGAA-3’ on the non-target strand. (D, E) cryo-EM reconstruction at 2.78 Å and cartoon representations of the IscB-ωRNA/target DNA complex.

Fig. 2.. Structural organization of the ωRNA and comparison to Cas9 crRNA-tracrRNA

(A) Schematic of ωRNA depicting secondary and tertiary interactions. Non-target strand, red; target strand, blue; guide RNA, orange. (B) Atomic model of ωRNA. (C) Close-up view depicting R-loop base pairing between guide RNA and target strand DNA. (D) Structural alignment of ωRNA and tracrRNA-crRNA in SpCas9 RNP showing conserved RNA structures in guide RNAs, P1 with SpCas9 tracrRNA-crRNA helix, J1 with SpCas9 tracrRNA stem loop 1, P3 pseudoknot with SpCas9 tracrRNA stem loop 2, and P5 with SpCas9 tracrRNA stem loop 3. Colored in black is the region of the ωRNA replaced by the REC lobe in Cas9.

Fig. 3.. Structural basis for TAM recognition and R-loop formation by IscB-ωRNA.

(A) TAM recognition and R-loop specification by domains of IscB. Color scheme is consistent with Fig. 1. (B) Close-up view of P1 interaction domain (P1D) linker residues recognizing TAM-2 basepair (target adjacent motif) from the DNA minor groove side. (C) Close-up view of the IscB TAM interaction domain (TID) making base-specific contacts from the DNA major groove side. (D) Close-up view of the bridge helix and P1D making contacts with the beginning portion of the DNA/RNA heteroduplex in the R-loop region. (E) Close-up view of the β-hairpin+linker domain specifying meandering the minor groove of the middle portion of the DNA/RNA heteroduplex. (F) Diagram of IscB contacts to TAM and DNA/RNA heteroduplex in the R-loop. Positioning of bridge helix domain separating the R-loop from the core of ωRNA in light blue. Green lines denote electrostatic contacts and brown lines denote hydrophobic contacts. TAM highlighted with purple box (ideal TAM sequence: 5’-NWRRNA-3’). guide RNA (orange), target strand DNA (blue), non-target strand DNA (red).

Figure 4.. Mechanistic dissection of RNA-guided DNA cleavage by IscB.

(A) 3.7 Å EM map and atomic model depicting the unlocked R-loop state. Color scheme is consistent with that in Fig. 1. (B) Focused view of DNA, guide RNA, and nuclease densities seen in the unlocked R-loop state. Note that NTS is blocked from entering the RuvC cleavage site by the anchor of HNH to RuvC. (C) 3.8 Å EM map and atomic model of the locked R-loop state. Alphafold predicted HNH domain structure (in green) is docked unambiguously into the EM density. Linker between HNH and RuvC domains can be seen interacting with the TAM-distal portion of the R-loop. (D) Focused view of HNH densities in the locked (active) state. Note that the NTS density is now allowed into the RuvC active site. (E) Close-up view of the HNH active site in the locked state. Catalytic metal ion (black) is seen coordinated to the TS substrate. A second metal ion is required for cleavage (ball with dash line). It is repelled from the active site by the phosphothioate modification in DNA. (F) Close-up view of the RuvC active site in the locked R-loop state. The coordinated catalytic metal ion (black) is seen contacting the backbone of the incoming NTS DNA. (G) Urea-PAGE showing time-resolved DNA cleavage. TS is cleaved by HNH prior to NTS cleavage by RuvC, supporting the unlocked/locked R-loop cleavage model. (H) Proposed mechanistic model explaining ordered strand cleavage by IscB. (I) Small RNA-seq of purified IscB-RNP, showing partial degradation of the guide RNA and a predictable cleavage site preceding stemloop P5. (J) Domain organization of wild-type and ΔPLMP IscB. (K) Urea-PAGE showing time-resolved DNA cleavage by IscB ΔPLMP.