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

Schuler, Gabriel; Hu, Chunyi; Ke, Ailong

doi:10.1126/science.abq7220

Cited by 53 publications

(39 citation statements)

References 28 publications

(50 reference statements)

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“…For example, the RECL in some, but not all clades of IscB, is larger than the corresponding linker region in IsrB and folds into a minimal secondary structure, whereas in YnpsCas9- To characterize in greater detail the minimization of the ωRNA as it evolved into cr/tracrRNAs, we compared the structure of DtIsrB ωRNA (DtRNA) with those of OgeuIscB ωRNA (OgRNA), CjCas9 single-guide RNA (CjRNA) and SpCas9 sgRNA in their protein/target DNA-bound states (Extended Data Fig. 9) [16][17][18] . On the basis of topology, location and secondary structure, we mapped DtRNA structural features (S1-4 and SL1-8) on other RNA species and named unidentified structural motifs as motifs 1-5 (M1-5).…”

Section: Discussionmentioning

confidence: 99%

“…To trace the protein domain evolution from IsrB to Cas9, we compared the structure of IsrB with the structure of one of the largest known IscBs (OgeuIscB) 17 , a distant relative of IsrB containing the HNH nuclease domain, and the predicted structure of YnpsCas9-1 (an early branching Cas9 of subtype II-D from Ga0315277_10040887 that is among the Cas9s most closely similar to IscB) 4 (Extended Data Fig. 8 ).…”

Section: Discussionmentioning

confidence: 99%

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Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA

Hirano

Kappel

Altae-Tran

et al. 2022

Nature

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RNA-guided systems, such as CRISPR–Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies1,2. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development3. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain4. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA

Hirano

Kappel

Altae-Tran

et al. 2022

Nature

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“…4a, b). This observation can explain why mutations in the PLMP motif reduced the IscB-mediated DNA cleavage 7,10 . The RuvC domain adopts an RNase H fold and the configuration of its catalytic residues (D61, E193, H340, and D343) is similar to that in Cas9 11,12 (Fig.…”

Section: Iscb Structurementioning

confidence: 96%

“…9 National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA. 10 Department of Chemistry and Biotechnology, Graduate School of Engineering, The University of Tokyo, Tokyo, Japan. 11 Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.…”

Section: Reporting Summarymentioning

confidence: 99%

Structure of the IscB–ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9

et al. 2022

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Transposon-encoded IscB family proteins are RNA-guided nucleases in the OMEGA (obligate mobile element-guided activity) system, and likely ancestors of the RNA-guided nuclease Cas9 in the type II CRISPR-Cas adaptive immune system. IscB associates with its cognate ωRNA to form a ribonucleoprotein complex that cleaves double-stranded DNA targets complementary to an ωRNA guide segment. Although IscB shares the RuvC and HNH endonuclease domains with Cas9, it is much smaller than Cas9, mainly due to the lack of the α-helical nucleic-acid recognition lobe. Here, we report the cryo-electron microscopy structure of an IscB protein from the human gut metagenome (OgeuIscB) in complex with its cognate ωRNA and a target DNA, at 2.6-Å resolution. This high-resolution structure reveals the detailed architecture of the IscB–ωRNA ribonucleoprotein complex, and shows how the small IscB protein assembles with the ωRNA and mediates RNA-guided DNA cleavage. The large ωRNA scaffold structurally and functionally compensates for the recognition lobe of Cas9, and participates in the recognition of the guide RNA–target DNA heteroduplex. These findings provide insights into the mechanism of the programmable DNA cleavage by the IscB–ωRNA complex and the evolution of the type II CRISPR-Cas9 effector complexes.

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“…Notably, a recent structural study shows that the IscB effector (commonly-acknowledged ancestor for type II Cas9 effectors) comprises an IscB nuclease monomer smaller than reported Cas9s and the ωRNA significantly larger than reported tracr-crRNA hybrids (Figure S11a). (Schuler et al, 2022) (which was not certified by peer review) is the author/funder. All rights reserved.…”

Section: A Hypothetical Evolution Trend Underlying Class 2 Crispr Eff...mentioning

confidence: 99%

The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation

Sun

Chen

et al. 2022

Preprint

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CRISPR-Cas modules serve as the adaptive nucleic-acid immune systems for prokaryotes, and provide versatile tools for nucleic-acid manipulation in various organisms. Here, we discovered a new miniature type V system, CRISPR-Cas pi (Cas12l) (~860 aa), from the environmental metagenome. Complexed with a large guide-RNA (~170 nt) comprising the tracrRNA and crRNA, Cas pi. (Cas12l) recognizes a unique 5' C-rich PAM for DNA cleavage under a broad range of biochemical conditions, and generates gene editing in mammalian cells. Cryo-EM study reveals a 'bracelet' architecture of Cas pi effector encircling the DNA target at 3.4-A resolution, substantially different from the canonical 'two-lobe' architectures of Cas12 and Cas9 nucleases. And the large guide-RNA serves as a 'two-arm' scaffold for effector assembly. Our study expands the knowledge of DNA targeting mechanisms by CRISPR effectors, and offers an efficient but compact platform for DNA manipulations.

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

Cited by 53 publications

References 28 publications

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA

Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA

Structure of the IscB–ωRNA ribonucleoprotein complex, the likely ancestor of CRISPR-Cas9

The compact Casπ (Cas12l) ‘bracelet’ provides a unique structural platform for DNA manipulation

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