Programmable type III-A CRISPR-Cas DNA targeting modules

• Published in: PloS one 2017-04, Vol.12 (4), p.e0176221
• Main Authors: Ichikawa, H Travis, Cooper, John C, Lo, Leja, Potter, Jason, Terns, Rebecca M, Terns, Michael P
• Format: Article
• Language: English
• Subjects: Ampicillin; Arabinose; Archaea; Assaying; Bacteria; Bacteriology; Biochemistry; Biodegradation; Biology; Biology and life sciences; Cellular apoptosis susceptibility protein; Chloramphenicol; Clustered Regularly Interspaced Short Palindromic Repeats; Colonies; Construction; CRISPR; CRISPR-Cas Systems; Crystal structure; Defense industry; Degradation; Deoxyribonucleic acid; Dilution; DNA; DNA - genetics; DNA structure; DNA topoisomerase IV; E coli; Engineering; Engineering and Technology; Escherichia coli - genetics; Exonuclease; Gene expression; Genes; Genetic aspects; Genetics; Genomes; Homology; Lactobacillus; Medicine and Health Sciences; Microbiology; Modules; Molecular biology; Nuclease; Nucleic acids; Nucleotide sequence; Phages; Physical Sciences; Physiological aspects; Plasmids; Prokaryotes; Proteins; Replication origins; Research and analysis methods; Ribonucleic acid; RNA; Staphylococcus; Transcription; Viruses
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0176221

The CRISPR-Cas systems provide invader defense in a wide variety of prokaryotes, as well as technologies for many powerful applications. The Type III-A or Csm CRISPR-Cas system is one of the most widely distributed across prokaryotic phyla, and cleaves targeted DNA and RNA molecules. In this work, we have constructed modules of Csm systems from 3 bacterial species and heterologously expressed the functional modules in E. coli. The modules include a Cas6 protein and a CRISPR locus for crRNA production, and Csm effector complex proteins. The expressed modules from L. lactis, S. epidermidis and S. thermophilus specifically eliminate invading plasmids recognized by the crRNAs of the systems. Characteristically, activation of plasmid targeting activity depends on transcription of the plasmid sequence recognized by the crRNA. Activity was not observed when transcription of the crRNA target sequence was blocked, or when the opposite strand or a non-target sequence was transcribed. Moreover, the Csm module can be programmed to recognize plasmids with novel target sequences by addition of appropriate crRNA coding sequences to the module. These systems provide a platform for investigation of Type III-A CRISPR-Cas systems in E. coli, and for introduction of programmable transcription-activated DNA targeting into novel organisms.