The Cas6e ribonuclease is not required for interference and adaptation by the E. coli type I-E CRISPR-Cas system.
Bottom Line: Type I CRISPR-Cas systems are composed of a multiprotein complex (Cascade) that, when bound to CRISPR RNA (crRNA), can recognize double-stranded DNA targets and recruit the Cas3 nuclease to destroy target-containing DNA.Here, we show that when mature unit-sized crRNAs are provided in a Cas6e-independent manner by transcription termination, the CRISPR-Cas system can function without Cas6e.The results should allow facile interrogation of various targets by type I-E CRISPR-Cas system in E. coli using unit-sized crRNAs generated by transcription.
Affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.Show MeSH
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Mentions: Cells carrying a cas6eH20A allele and expressing crRNA from natural CRISPR arrays are incapable of primed adaptation (18). We followed CRISPR adaptation in cells harboring pG8_crRNA transformed with compatible plasmid containing protospacer. Individual transformants were grown at conditions of induction of cas genes in the absence of ampicillin in the medium, thus allowing the protospacer plasmid loss. After overnight growth, culture aliquots were analyzed by PCR to monitor CRISPR array expansion. KD263 cells transformed with protospacer plasmid were used as a control. The results are presented in Figure 6A. As expected, adaptation was observed in KD263 cells (18,32). Similar levels of adaptation were also observed in KD390 and H20A cells transformed with protospacer plasmid. A somewhat reduced adaptation level was detected in Δcas6e cultures. No adaptation was observed in cells harboring a plasmid without a protospacer. Therefore, unit-sized crRNA generated without Cas6e processing appears to support primed adaptation and the endoribonucleolytic activity of Cas6e (and in fact the Cas6e protein itself) is dispensable in this context.
Affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.