Abstract
The Type I-F CRISPR-mediated (clustered regularly interspaced short palindromic repeats) adaptive immune system in Pseudomonas aeruginosa consists of two CRISPR loci and six CRISPR-associated (cas) genes. Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA (crRNA) into a 350-kDa ribonucleoprotein called the Csy complex. Here, we show that foreign nucleic acid recognition by the Csy complex proceeds through sequential steps, initiated by detection of two consecutive guanine–cytosine base pairs (G–C/G–C) located adjacent to the complementary DNA target. We show that this motif, called the PAM (protospacer adjacent motif), must be double-stranded and that single-stranded PAMs do not provide significant discriminating power. Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for crRNA-guided binding to a target sequence. Together, these data indicate that the Csy complex first interacts with G–C/G–C base pairs and then samples adjacent target sequences for complementarity to the crRNA guide.
Highlights
Bacteria and archaea have evolved sophisticated nucleic acid-based adaptive immune systems to defend against exogenous genetic elements like viruses and plasmids [1,2,3,4,5,6]
Foreign DNA surveillance is performed by a complex of Cas proteins (Csy1–4) that assemble with a CRISPR RNA into a 350-kDa ribonucleoprotein called the Csy complex
Binding assays performed with G–C/G–C-rich competitor sequences indicate that the Csy complex interacts directly with this dinucleotide motif, and kinetic analyses reveal that recognition of a G–C/G–C motif is a prerequisite for CRISPR RNA (crRNA)-guided binding to a target sequence
Summary
Bacteria and archaea have evolved sophisticated nucleic acid-based adaptive immune systems to defend against exogenous genetic elements like viruses and plasmids [1,2,3,4,5,6]. Immunity is acquired by integrating short segments of foreign DNA into one end of the host encoded CRISPR locus (clustered regularly interspaced short palindromic repeats) These foreign sequences, called spacers, are flanked by short repeat sequences, creating the repeat–spacer–repeat pattern that is characteristic of CRISPR-mediated immune systems. While some Type III systems target RNA [8,9,10,11,12], the Types I and II systems target and destroy invading DNA [13,14,15,16,17,18,19] All of these crRNA-guided surveillance complexes must locate target sequences on a time scale that affords protection from rapidly replicating phages, and CRISPR systems that target DNA must be able to reliably distinguish complementary spacer sequences in the host CRISPR locus (self) from identical protospacer sequences in the invading DNA target (non-self). The PAM is only found next to complementary protospacer targets in foreign DNA, and is absent from repeat sequences that flank complementary spacer sequences in the host CRISPR locus
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