CRISPR RNA-Dependent Binding and Cleavage of Endogenous RNAs by the Campylobacter jejuni Cas9.

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 Campylobacter jejuni 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 in vitro. These findings suggest that C. jejuni Cas9 is a promiscuous nuclease that can coordinately target both DNA and RNA.

[1]  Max J. Kellner,et al.  RNA editing with CRISPR-Cas13 , 2017, Science.

[2]  J. Vogel,et al.  Deep Sequencing Analysis of Small Noncoding RNA and mRNA Targets of the Global Post-Transcriptional Regulator, Hfq , 2008, PLoS genetics.

[3]  Oscar A. Negrete,et al.  RNA-dependent RNA targeting by CRISPR-Cas9 , 2018, eLife.

[4]  Yan Zhang,et al.  DNase H Activity of Neisseria meningitidis Cas9. , 2015, Molecular cell.

[5]  Alex E. Lash,et al.  Gene Expression Omnibus: NCBI gene expression and hybridization array data repository , 2002, Nucleic Acids Res..

[6]  R. Reinhardt,et al.  The CsrA-FliW network controls polar localization of the dual-function flagellin mRNA in Campylobacter jejuni , 2016, Nature Communications.

[7]  J. Doudna,et al.  CRISPR-Cas9 Structures and Mechanisms. , 2017, Annual review of biophysics.

[8]  Kira S. Makarova,et al.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems , 2013, Nucleic acids research.

[9]  Kristin Reiche,et al.  The primary transcriptome of the major human pathogen Helicobacter pylori , 2010, Nature.

[10]  Takanori Nakane,et al.  Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems. , 2017, Molecular cell.

[11]  Lucas B. Harrington,et al.  Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. , 2015, Molecular cell.

[12]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[13]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[14]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[15]  Eunji Kim,et al.  In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni , 2017, Nature Communications.

[16]  Sita J. Saunders,et al.  An updated evolutionary classification of CRISPR–Cas systems , 2015, Nature Reviews Microbiology.

[17]  Konrad U. Förstner,et al.  READemption - a tool for the computational analysis of deep-sequencing-based transcriptome data , 2014, Bioinform..

[18]  Gene W. Yeo,et al.  Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9 , 2017, Cell.

[19]  Peter C. Fineran,et al.  CRISPR–Cas systems: beyond adaptive immunity , 2014, Nature Reviews Microbiology.

[20]  H. Margalit,et al.  Evolutionary patterns of Escherichia coli small RNAs and their regulatory interactions , 2014, RNA.

[21]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[22]  U. Alon,et al.  A comprehensive library of fluorescent transcriptional reporters for Escherichia coli , 2006, Nature Methods.

[23]  Peter F. Stadler,et al.  Fast Mapping of Short Sequences with Mismatches, Insertions and Deletions Using Index Structures , 2009, PLoS Comput. Biol..

[24]  Gene W. Yeo,et al.  Applications of Cas9 as an RNA-programmed RNA-binding protein. , 2015, BioEssays : news and reviews in molecular, cellular and developmental biology.

[25]  M. Peterka,et al.  Complete Genome Sequences of Group III Campylobacter Bacteriophages PC5 and PC14 , 2016, Genome Announcements.

[26]  Eric S. Lander,et al.  C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector , 2016, Science.

[27]  Qijing Zhang,et al.  Identification and characterisation of new Campylobacter group III phages of animal origin. , 2014, FEMS microbiology letters.

[28]  Benjamin L. Oakes,et al.  Programmable RNA recognition and cleavage by CRISPR/Cas9 , 2014, Nature.

[29]  Luciano A. Marraffini,et al.  CRISPR-Cas immunity in prokaryotes , 2015, Nature.

[30]  D. Burstein,et al.  RNA Targeting by Functionally Orthogonal Type VI-A CRISPR-Cas Enzymes. , 2017, Molecular cell.

[31]  Conrad Steenberg,et al.  NUPACK: Analysis and design of nucleic acid systems , 2011, J. Comput. Chem..

[32]  Kay Nieselt,et al.  High-Resolution Transcriptome Maps Reveal Strain-Specific Regulatory Features of Multiple Campylobacter jejuni Isolates , 2013, PLoS genetics.

[33]  R. Barrangou,et al.  Expanding the CRISPR Toolbox: Targeting RNA with Cas13b. , 2017, Molecular cell.

[34]  David S. Weiss,et al.  A CRISPR-CAS System Mediates Bacterial Innate Immune Evasion and Virulence , 2013, Nature.

[35]  J. Vogel,et al.  Experimental tools to identify RNA-protein interactions in Helicobacter pylori , 2012, RNA biology.

[36]  David C. Norris,et al.  Integrated genome browser: visual analytics platform for genomics , 2015, bioRxiv.

[37]  Qian Wang,et al.  GFOLD: a generalized fold change for ranking differentially expressed genes from RNA-seq data , 2012, Bioinform..

[38]  Chase L. Beisel,et al.  Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. , 2016, Molecular cell.

[39]  Jennifer A. Doudna,et al.  Programmable RNA Tracking in Live Cells with CRISPR/Cas9 , 2016, Cell.

[40]  Ibtissem Grissa,et al.  The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats , 2007, BMC Bioinformatics.

[41]  H. Endtz,et al.  A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion , 2014, Proceedings of the National Academy of Sciences.

[42]  Jörg Vogel,et al.  Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. , 2013, Molecular cell.

[43]  Xiao-Hui Zhang,et al.  Off-target Effects in CRISPR/Cas9-mediated Genome Engineering , 2015, Molecular therapy. Nucleic acids.

[44]  P. Hsu,et al.  Methods for Optimizing CRISPR-Cas9 Genome Editing Specificity. , 2016, Molecular cell.

[45]  Aviv Regev,et al.  RNA targeting with CRISPR–Cas13 , 2017, Nature.

[46]  Aviv Regev,et al.  Nucleic acid detection with CRISPR-Cas13a/C2c2 , 2017, Science.

[47]  Chase L. Beisel,et al.  Deciphering, Communicating, and Engineering the CRISPR PAM. , 2017, Journal of molecular biology.

[48]  Charles Elkan,et al.  Fitting a Mixture Model By Expectation Maximization To Discover Motifs In Biopolymer , 1994, ISMB.

[49]  E. C. Soo,et al.  Campylobacter jejuni Glycosylation Island Important in Cell Charge, Legionaminic Acid Biosynthesis, and Colonization of Chickens , 2009, Infection and Immunity.

[50]  Sergey A. Shmakov,et al.  Cas13b is a Type VI-B CRISPR-associated RNA-Guided RNase differentially regulated by accessory proteins Csx27 and Csx28 , 2016, bioRxiv.

[51]  Adam M. Phillippy,et al.  Interactive metagenomic visualization in a Web browser , 2011, BMC Bioinformatics.

[52]  Kira S. Makarova,et al.  Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems , 2016, Science.

[53]  Eugene V Koonin,et al.  Diversity, classification and evolution of CRISPR-Cas systems. , 2017, Current opinion in microbiology.

[54]  Yuquan Wei,et al.  Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity , 2016, Cell Research.

[55]  David S. Weiss,et al.  Cas9-mediated targeting of viral RNA in eukaryotic cells , 2015, Proceedings of the National Academy of Sciences.

[56]  D. G. Gibson,et al.  Enzymatic assembly of DNA molecules up to several hundred kilobases , 2009, Nature Methods.

[57]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[58]  Nora C. Pyenson,et al.  Type III CRISPR-Cas systems: when DNA cleavage just isn't enough. , 2017, Current opinion in microbiology.

[59]  L. Randau,et al.  Commentary: Type I CRISPR-Cas targets endogenous genes and regulates virulence to evade mammalian host immunity , 2017, Front. Microbiol..

[60]  G. O’Toole,et al.  Interaction between Bacteriophage DMS3 and Host CRISPR Region Inhibits Group Behaviors of Pseudomonas aeruginosa , 2008, Journal of bacteriology.

[61]  Chase L. Beisel,et al.  Programmable Removal of Bacterial Strains by Use of Genome-Targeting CRISPR-Cas Systems , 2014, mBio.

[62]  Jason Hinds,et al.  Comparative phylogenomics of the food-borne pathogen Campylobacter jejuni reveals genetic markers predictive of infection source. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[63]  H. Endtz,et al.  A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain–Barré syndrome , 2012, European Journal of Clinical Microbiology & Infectious Diseases.