Transcription Termination and Antitermination of Bacterial CRISPR Arrays

Abstract

A hallmark of CRISPR-Cas immunity systems is the CRISPR array, a genomic locus consisting of short, repeated sequences (“repeats”) interspersed with short, variable sequences (“spacers”). CRISPR arrays are transcribed and processed into individual CRISPR RNAs (crRNAs) that each include a single spacer, and direct Cas proteins to complementary sequence in invading nucleic acid. Most bacterial CRISPR array transcripts are unusually long for untranslated RNA, suggesting the existence of mechanisms to prevent premature transcription termination by Rho, a conserved bacterial transcription termination factor that rapidly terminates untranslated RNA. We show that CRISPR arrays of Salmonella Typhimurium are protected from Rho by the Nus factor anti-termination complex, and we provide evidence that this is an evolutionarily ancient mechanism to facilitate complete transcription of bacterial CRISPR arrays.

A multiplex CRISPR interference tool for virulence gene interrogation in an intracellular pathogen

“…Our data reiterate that this is an advantageous strategy as the earliest spacers in the array are the most likely to be transcribed, and therefore, will produce the greatest quantity of protective crRNAs for an ongoing viral attack. Several bacterial CRISPR systems appear to have overcome the limitation of promoter-driven transcription processivity by inhibiting transcription termination through acquiring Nus factorbinding boxA sequences upstream of their arrays (39). Adding the boxA sequence to the synthetic array also presumably increased transcription processivity of our synthetic constructs, as genes targeted by crRNAs encoded by spacers in more distal positions were now efficiently silenced (Figure 4).…”

“…Stringer et. al recently discovered boxA elements to be evolutionarily conserved upstream of naturally occurring CRISPR arrays where they appear to promote expression of downstream spacers within long arrays (39). To investigate whether Rho-dependent transcriptional termination had prevented our CRISPRi platform from achieving maximum gene silencing from distal spacer positions, knowing that L. pneumophila does encode at least one member of the Nus complex (39), we explored if incorporation of boxA elements could overcome this limitation.…”