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  • 1
    Language: English
    In: Molecular Cell, 01 March 2018, Vol.69(5), pp.893-905.e7
    Description: Cas9 nucleases naturally utilize CRISPR RNAs (crRNAs) to silence foreign double-stranded DNA. While recent work has shown that some Cas9 nucleases can also target RNA, RNA recognition has required nuclease modifications or accessory factors. Here, we show that the Cas9 (CjCas9) can bind and cleave complementary endogenous mRNAs in a crRNA-dependent manner. Approximately 100 transcripts co-immunoprecipitated with CjCas9 and generally can be subdivided through their base-pairing potential to the four crRNAs. A subset of these RNAs was cleaved around or within the predicted binding site. Mutational analyses revealed that RNA binding was crRNA and tracrRNA dependent and that target RNA cleavage required the CjCas9 HNH domain. We further observed that RNA cleavage was PAM independent, improved with greater complementarity between the crRNA and the RNA target, and was programmable . These findings suggest that Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA. CRISPR-Cas9 nucleases typically target double-stranded DNA targets. Dugar et al. show that one of the smallest Cas9 proteins (from ) can also target endogenous RNAs. Targeting occurred through imperfect complementarity with native crRNA guides and cleavage by Cas9’s HNH domain.
    Keywords: Crispr ; Cas9 ; Crrna ; RNA Cleavage ; Campylobacter Jejuni ; Post-Transcriptional Regulation ; Non-Coding RNA ; Genome Editing ; RIP-Seq ; RNA Binding Proteins ; Biology
    ISSN: 1097-2765
    E-ISSN: 1097-4164
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  • 2
    Language: English
    In: Molecular Cell, 2008, Vol.32(6), pp.827-837
    Description: Small noncoding RNAs (sRNAs) have predominantly been shown to repress bacterial mRNAs by masking the Shine-Dalgarno (SD) or AUG start codon sequence, thereby preventing 30S ribosome entry and, consequently, translation initiation. However, many recently identified sRNAs lack obvious SD and AUG complementarity, indicating that sRNA-mediated translational control could also take place at other mRNA sites. We report that RybB sRNA represses mRNA translation by pairing with the 5′ coding region. Results of systematic antisense interference with 30S binding to and unrelated mRNAs suggest that sRNAs can act as translational repressors by sequestering sequences within the mRNA down to the fifth codon, even without SD and AUG start codon pairing. This “five codon window” for translational control in the 5′ coding region of mRNA not only has implications for sRNA target predictions but might also apply to -regulatory systems such as RNA thermosensors and riboswitches.
    Keywords: RNA ; Microbio ; Biology
    ISSN: 1097-2765
    E-ISSN: 1097-4164
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