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Nat Commun. 2019 Jul 5;10(1):3001. doi: 10.1038/s41467-019-10780-2.

Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble.

Liu TY1,2, Liu JJ1,2,3, Aditham AJ4, Nogales E1,2,3,5, Doudna JA6,7,8,9,10,11,12.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA.
2
California Institute for Quantitative Biosciences, Berkeley, CA, 94720, USA.
3
Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA.
4
Department of Chemical & Biomolecular Engineering, University of California, Berkeley, CA, 94720, USA.
5
Howard Hughes Medical Institute, University of California, Berkeley, CA, 94720, USA.
6
Department of Molecular and Cell Biology, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
7
California Institute for Quantitative Biosciences, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
8
Molecular Biophysics & Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
9
Howard Hughes Medical Institute, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
10
Gladstone Institutes, San Francisco, CA, 94158, USA. doudna@berkeley.edu.
11
Innovative Genomics Institute, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.
12
Department of Chemistry, University of California, Berkeley, CA, 94720, USA. doudna@berkeley.edu.

Abstract

Type III-A CRISPR-Cas systems are prokaryotic RNA-guided adaptive immune systems that use a protein-RNA complex, Csm, for transcription-dependent immunity against foreign DNA. Csm can cleave RNA and single-stranded DNA (ssDNA), but whether it targets one or both nucleic acids during transcription elongation is unknown. Here, we show that binding of a Thermus thermophilus (T. thermophilus) Csm (TthCsm) to a nascent transcript in a transcription elongation complex (TEC) promotes tethering but not direct contact of TthCsm with RNA polymerase (RNAP). Biochemical experiments show that both TthCsm and Staphylococcus epidermidis (S. epidermidis) Csm (SepCsm) cleave RNA transcripts, but not ssDNA, at the transcription bubble. Taken together, these results suggest that Type III systems primarily target transcripts, instead of unwound ssDNA in TECs, for immunity against double-stranded DNA (dsDNA) phages and plasmids. This reveals similarities between Csm and eukaryotic RNA interference, which also uses RNA-guided RNA targeting to silence actively transcribed genes.

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