Key result
Providing mature crRNAs via transcription termination enables E. coli CRISPR-Cas function without Cas6e.
Why the study?
The role of Cas6e ribonuclease in interference and adaptation by the Escherichia coli type I-E CRISPR-Cas system was unclear.
The study demonstrates that the E. coli type I-E CRISPR-Cas system can function without the Cas6e ribonuclease if mature crRNAs are provided via transcription termination, allowing for easier target interrogation.
Shortened crRNA spacers yield functional Cascade complexes; leaves open whether this extends CRISPR toolkits beyond prokaryotes.
CRISPR-Cas are small RNA-based adaptive prokaryotic immunity systems protecting cells from foreign DNA or RNA. 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. In the Escherichia coli type I-E CRISPR-Cas system, crRNAs are generated upon transcription of CRISPR arrays consisting of multiple palindromic repeats and intervening spacers through the function of Cas6e endoribonuclease, which cleaves at specific positions of repeat sequences of the CRISPR array transcript. Cas6e is also a component of Cascade. 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.
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Semenova et al. (2015) studied this question. Cas6e-independent provision of mature unit-sized crRNAs was evaluated on CRISPR-Cas system function (interference and adaptation). The E. coli type I-E CRISPR-Cas system can function without the Cas6e endoribonuclease when mature unit-sized crRNAs are provided by transcription termination.
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