Abstract
The CRISPR–Cas system for prokaryotic adaptive immunity provides RNA-mediated protection from viruses and mobile genetic elements. Adaptation is dependent on the Cas1 and Cas2 proteins along with varying accessory proteins. Here we analyse the process in Sulfolobus solfataricus, showing that while Cas1 and Cas2 catalyze spacer integration in vitro, host factors are required for specificity. Specific integration also requires at least 400 bp of the leader sequence, and is dependent on the presence of hydrolysable ATP, suggestive of an active process that may involve DNA remodelling. Specific spacer integration is associated with processing of prespacer 3′ ends in a PAM-dependent manner. This is reflected in PAM-dependent processing of prespacer 3′ ends in vitro in the presence of cell lysate or the Cas4 nuclease, in a reaction consistent with PAM-directed binding and protection of prespacer DNA. These results highlight the diverse interplay between CRISPR–Cas elements and host proteins across CRISPR types.
Highlights
CRISPR–Cas systems are present in around half of bacterial and 90% of archaeal genomes sequenced to date and form an adaptive immune system important in defence against invasion by foreign nucleic acids
Deletion of 100 bp sections internal to the leader abolished integration. These results indicate that the full length of the long leader sequences found in systems such as S. solfataricus are important for specific integration, in marked contrast to the situation in types I-E, I-F and II-A
The CRISPR–Cas system of S. solfataricus is one of the most complex studied to date, with multiple CRISPR repeats and loci, adaptation modules and effector complexes [44]
Summary
CRISPR–Cas systems are present in around half of bacterial and 90% of archaeal genomes sequenced to date and form an adaptive immune system important in defence against invasion by foreign nucleic acids. Key to CRISPR– Cas immunity is the ability to adapt to new threats by incorporating short segments of foreign DNA, called spacers, into the CRISPR array of the host. These spacers constitute immunological memories that are used by CRISPRassociated (Cas) proteins to mount sequence-specific defence on subsequent infection. The process of acquiring new spacers is termed Adaptation and can be divided into two main stages: firstly, the generation and capture of a prespacer by Cas, Cas (and potentially other) proteins and secondly, the docking of this nucleoprotein complex at the leader:repeat site, leading to integration of the new spacer by transesterification.
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