A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition

Mohr, Georg; Silas, Sukrit; Stamos, Jennifer L.; Makarova, Kira S.; Markham, Laura M.; Yao, Jun; Lucas-Elı́o, Patricia; Sánchez-Amat, Antonio; Fire, Andrew; Koonin, Eugene V.; Lambowitz, Alan M.

doi:10.1016/j.molcel.2018.09.013

Cited by 26 publications

(45 citation statements)

References 56 publications

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“…Finally, the structure of the Cas6 domain closely resembles those of previous Cas6 structures 34,36,[45][46][47][48] ( Supplementary Fig. 3c).…”

Section: Architecture Of the Cas6-rt-cas1-cas2 Complexsupporting

confidence: 77%

“…3c). When compared directly to the structure of the Cas6 domain from the Marinomonas mediterranea (MMB-1) Cas6-RT-Cas1 protein 34 , the two structures align with RMSD of 2.4 Å, with the greatest variance in the β-strand of the C-terminal RNA recognition motif (RRM) fold, perhaps due to the subsequent connection to the RT domain, absent in the MMB-1 Cas6 structure ( Supplementary Fig. 3d).…”

Section: Architecture Of the Cas6-rt-cas1-cas2 Complexmentioning

confidence: 99%

“…Notably, many of these RT-Cas1 fusion proteins include an N-terminal Cas6 domain 24,26,34 , which processes CRISPR transcripts into mature crRNAs [35][36][37] . A recent study showed that the Cas6 domain of a Cas6-RT-Cas1 fusion is required for RNA spacer acquisition and co-evolves with the RT domain 34 . However, the molecular basis for functional coordination between Cas1, RT and Cas6 remains unknown.…”

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confidence: 99%

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Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

Wang

Hoel

Al-Shayeb

et al. 2020

Preprint

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CRISPR-Cas systems provide adaptive immunity in bacteria and archaea by targeting foreign DNA for destruction using CRISPR RNA-guided enzymes. CRISPR immunity begins with integration of foreign sequences into the host CRISPR genomic locus, followed by transcription and maturation of CRISPR RNAs. In a few CRISPR systems, the Cas1 integrase and a Cas6 nuclease are fused to a reverse transcriptase that enables viral sequence acquisition from both DNA and RNA sources. To determine how these components work together, we determined a 3.7 Å resolution cryo-EM structure of a Cas6-RT-Cas1 protein complexed with Cas2, a subunit of the CRISPR integrase. The structure and accompanying mutagenesis experiments provide evidence of bidirectional crosstalk between the Cas1 and RT active sites and unidirectional crosstalk from Cas6 to the Cas1 and RT active sites. Together, these findings suggest regulated structural rearrangements that may coordinate the complex's different enzymatic activities.

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“…Finally, the structure of the Cas6 domain closely resembles those of previous Cas6 structures 34,36,[45][46][47][48] ( Supplementary Fig. 3c).…”

Section: Architecture Of the Cas6-rt-cas1-cas2 Complexsupporting

confidence: 77%

Section: Architecture Of the Cas6-rt-cas1-cas2 Complexmentioning

confidence: 99%

mentioning

confidence: 99%

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Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

Wang

Hoel

Al-Shayeb

et al. 2020

Preprint

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“…Our work provides the temporal resolution to dissect the kinetic framework governing prespacer biogenesis, integration, and incorporation processes. It establishes the foundation to further understand spacer acquisitions processes that involve specialized processing factors, such as Cas3 and Cas4 nucleases 27 – 29 , 36 , 41 – 43 and reverse transcriptase 44 – 47 . The most important conceptual advance of this study is the realization that an efficient mechanism to resolve the post-synaptic complex is essential to maintain robust CRISPR-Cas immunity.…”

Section: Discussionmentioning

confidence: 99%

Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

Budhathoki

Xiao

Schuler

et al. 2020

Nat Struct Mol Biol

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Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the Enterococcus faecalis ( Efa ) Cas1–Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. Efa Cas1–Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes Efa Cas1–Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex has a surprisingly short mean lifetime. Remarkably, transcription efficiently resolves the postsynaptic complex and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.

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“…Interestingly, the CRISPR-Cas systems encoding RTs usually correspond to a subset of type III systems [15][16][17][18] from all known subtypes (III-A, III-B, III-C and III-D). Attention was recently focused on this association by the discovery of RT-mediated spacer acquisition from RNA molecules in a type III-B system from Marinomonas mediterranea [15,28], and the more recent description of an RT-Cas1 fusion from Fusicatenibacter saccharivorans in the so-called 'Record-seq' method, making it possible to make transcriptome-scale molecular recordings in cell populations [29].…”

Section: Introductionmentioning

confidence: 99%

Multiple origins of reverse transcriptases linked to CRISPR-Cas systems

et al. 2019

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Prokaryotic genomes harbour a plethora of uncharacterized reverse transcriptases (RTs). RTs phylogenetically related to those encoded by group-II introns have been found associated with type III CRISPR-Cas systems, adjacent or fused at the C-terminus to Cas1. It is thought that these RTs may have a relevant function in the CRISPR immune response mediating spacer acquisition from RNA molecules. The origin and relationships of these RTs and the ways in which the various protein domains evolved remain matters of debate. We carried out a large survey of annotated RTs in databases (198,760 sequences) and constructed a large dataset of unique representative sequences (9,141). The combined phylogenetic reconstruction and identification of the RTs and their various protein domains in the vicinity of CRISPR adaptation and effector modules revealed three different origins for these RTs, consistent with their emergence on multiple occasions: a larger group that have evolved from group-II intron RTs, and two minor lineages that may have arisen more recently from Retron/retron-like sequences and Abi-P2 RTs, the latter associated with type I-C systems. We also identified a particular group of RTs associated with CRISPR-cas loci in clade 12, fused C-terminally to an archaeo-eukaryotic primase (AEP), a protein domain (AE-Prim_S_like) forming a particular family within the AEP proper clade. Together, these data provide new insight into the evolution of CRISPR-Cas/RT systems.

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A Reverse Transcriptase-Cas1 Fusion Protein Contains a Cas6 Domain Required for Both CRISPR RNA Biogenesis and RNA Spacer Acquisition

Cited by 26 publications

References 56 publications

Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

Structural coordination between active sites of a Cas6-reverse transcriptase-Cas1—Cas2 CRISPR integrase complex

Real-time observation of CRISPR spacer acquisition by Cas1–Cas2 integrase

Multiple origins of reverse transcriptases linked to CRISPR-Cas systems

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