title: The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.
abstract: The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.
authors.affiliation: Institute of Biotechnology, Vilnius University, Graiciuno 8, LT-02241 Vilnius, Lithuania.
authors.forename: Rimantas
authors.initials: R
authors.lastname: Sapranauskas
authors.forename: Giedrius
authors.initials: G
authors.lastname: Gasiunas
authors.forename: Christophe
authors.initials: C
authors.lastname: Fremaux
authors.forename: Rodolphe
authors.initials: R
authors.lastname: Barrangou
authors.forename: Philippe
authors.initials: P
authors.lastname: Horvath
authors.forename: Virginijus
authors.initials: V
authors.lastname: Siksnys
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics* / Q000276:immunology / Q000821:virology; D007109:Immunity; D008969:Molecular Sequence Data; D009154:Mutation; D059372:Nucleotide Motifs; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012091:Repetitive Sequences, Nucleic Acid; D048249:Streptococcus thermophilus / Q000235:genetics*; D014169:Transformation, Bacterial
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial