Spatiotemporal Control of Type III-A CRISPR-Cas Immunity: Coupling DNA Degradation with the Target RNA Recognition.
Streptococcus thermophilus (St) type III-A CRISPR-Cas system restricts MS2 RNA phage and cuts RNA in vitro. However, the CRISPR array spacers match DNA phages, raising the question: does the St CRISPR-Cas system provide immunity by erasing phage mRNA or/and by eliminating invading DNA? We show that it does both. We find that (1) base-pairing between crRNA and target RNA activates single-stranded DNA (ssDNA) degradation by StCsm; (2) ssDNase activity is confined to the HD-domain of Cas10; (3) target RNA cleavage by the Csm3 RNase suppresses Cas10 DNase activity, ensuring temporal control of DNA degradation; and (4) base-pairing between crRNA 5'-handle and target RNA 3'-flanking sequence inhibits Cas10 ssDNase to prevent self-targeting. We propose that upon phage infection, crRNA-guided StCsm binding to the emerging transcript recruits Cas10 DNase to the actively transcribed phage DNA, resulting in degradation of both the transcript and phage DNA, but not the host DNA.
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
Migle
M
Kazlauskiene
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Electronic address: tamulaitis@ibt.lt.
Gintautas
G
Tamulaitis
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
Georgij
G
Kostiuk
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania.
Česlovas
Č
Venclovas
Institute of Biotechnology, Vilnius University, Graiciuno 8, Vilnius 02241, Lithuania. Electronic address: siksnys@ibt.lt.
Virginijus
V
Siksnys
D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000276:immunology; D004269:DNA, Bacterial / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004279:DNA, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology / Q000821:virology; D054884:Host-Pathogen Interactions; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D059367:RNA Cleavage; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D048249:Streptococcus thermophilus / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000821:virology; D013997:Time Factors
D064130:CRISPR-Associated Proteins; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D012333:RNA, Messenger; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase