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Proc Natl Acad Sci U S A. 2018 Mar 27;115(13):3368-3373. doi: 10.1073/pnas.1717725115. Epub 2018 Mar 12.

Programmable RNA recognition using a CRISPR-associated Argonaute.

Lapinaite A1, Doudna JA1,2,3,4,5, Cate JHD6,2,5.

Author information

1
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720.
2
Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
3
Howard Hughes Medical Institute, University of California, Berkeley, CA 94720.
4
Innovative Genomics Institute, University of California, Berkeley, CA 94720.
5
Department of Chemistry, University of California, Berkeley, CA 94720.
6
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720; jcate@lbl.gov.

Abstract

Argonaute proteins (Agos) are present in all domains of life. Although the physiological function of eukaryotic Agos in regulating gene expression is well documented, the biological roles of many of their prokaryotic counterparts remain enigmatic. In some bacteria, Agos are associated with CRISPR (clustered regularly interspaced short palindromic repeats) loci and use noncanonical 5'-hydroxylated guide RNAs (gRNAs) for nucleic acid targeting. Here we show that using 5-bromo-2'-deoxyuridine (BrdU) as the 5' nucleotide of gRNAs stabilizes in vitro reconstituted CRISPR-associated Marinitoga piezophila Argonaute-gRNA complexes (MpAgo RNPs) and significantly improves their specificity and affinity for RNA targets. Using reconstituted MpAgo RNPs with 5'-BrdU-modified gRNAs, we mapped the seed region of the gRNA and identified the nucleotides of the gRNA that play the most significant role in targeting specificity. We also show that these MpAgo RNPs can be programmed to distinguish between substrates that differ by a single nucleotide, using permutations at the sixth and seventh positions in the gRNA. Using these specificity features, we employed MpAgo RNPs to detect specific adenosine-to-inosine-edited RNAs in a complex mixture. These findings broaden our mechanistic understanding of the interactions of Argonautes with guide and substrate RNAs, and demonstrate that MpAgo RNPs with 5'-BrdU-modified gRNAs can be used as a highly specific RNA-targeting platform to probe RNA biology.

KEYWORDS:

Argonaute; CRISPR; RNA editing; inosine; small noncoding RNA

PMID:
29531059
PMCID:
PMC5879674
DOI:
10.1073/pnas.1717725115
[Indexed for MEDLINE]
Free PMC Article

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