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Research Teams Reveal Possible Roles of Cas4 in CRISPR Adaptation

NEW YORK (GenomeWeb) – Of all the Cas proteins researchers have discovered associated with the CRISPR genome editing system in bacteria, Cas4 has remained somewhat mysterious as researchers have yet to discover what its purpose really is.

Now, however, two research teams have independently published studies in which they've established models for possible roles Cas4 could play in the CRISPR editing process.

In one study published in Molecular Cell today, researchers from Iowa State University and the University of Texas at Austin wrote that Cas4 proteins are thought to participate in the acquisitions of spacers — short segments of foreign DNA that are integrated by CRISPR systems into the host CRISPR locus to provide molecular memory of infection. In their study, the researchers also showed that type I-C Cas4 in the Bacillus halodurans bacterium is required for efficient prespacer processing prior to Cas1-Cas2-mediated integration.

"Cas4 recognizes [protospacer adjacent motif] PAM sequences within the prespacer and prevents integration of unprocessed prespacers, ensuring that only functional spacers will be integrated into the CRISPR array," the authors wrote. "Our results reveal the critical role of Cas4 in maintaining fidelity during CRISPR adaptation, providing a structural and mechanistic model for prespacer processing and integration."

Through several biochemical experiments, the team found that Cas4 cuts precursors at specific locations through endonucleolytic cleavage, suggesting that Cas4 associates with the Cas1-Cas2 complex, and that the complex positions the 3' overhangs in the Cas4 active site to dictate the exact cut sites.

Further, when the researchers conducted structural studies of the Cas4-Cas1 complex, they found that the architecture may be mutually exclusive with formation of the Cas1-Cas2 complex structure observed in other subtypes.

"It is possible that type I-C Cas1-Cas2 adopts a different overall conformation. Alternatively, it is possible that the Cas4-Cas1 complex sequesters Cas1, preventing it from forming a productive Cas1-Cas2 complex for integrating dsDNA substrates. Thus, these competing structures could provide a regulatory mechanism for the adaptation stage of CRISPR immunity," they wrote. "Future structural work will be required to determine how the Cas4-Cas1 structure transitions to the overall adaptation complex."

Their mutagenesis analyses then showed that the Cas4 active site is necessary and sufficient for efficient prespacer processing, while the Cas1 active site can catalyze low levels of cleavage in the absence of Cas4. This suggested that long single-stranded 3' overhangs may shuttle between the Cas4 and Cas1 active sites and may be preferentially bound by Cas4 until cleavage occurs, preventing either cleavage or integration by the Cas1 active site. Further, they observed integration following prespacer processing in the presence of Cas4, while integration of unprocessed prespacers was much more prevalent in the absence of Cas4.

In addition to enhancing prespacer cleavage, Cas4 provides a "fidelity check" to ensure that prespacers are only integrated following removal of the PAM sequence, the researchers found. "Integration prior to processing is likely to result in a spacer targeting a non-PAM region. Thus, our results suggest that Cas4-dependent processing is critical for maintaining a fully functional CRISPR array, without the addition of defective spacers through aberrant integration prior to prespacer processing," they wrote.

In another study, published today in Cell Reports, researchers in Germany and the Netherlands introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of the Synechocystis sp. 6803 bacterium into Escherichia coli. They found that the cas4 gene is strictly required for the selection of targets with PAMs conferring I-D CRISPR interference in the native host Synechocystis.

"We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements," the authors wrote.

In this study, the researchers largely focused on the question of how bacteria select spacers that confer functional CRISPR interference. Through a series of experiments, they found that the Cas4 protein present in type I, II, and V systems helps to integrate spacers targeting DNA sequences with PAMs that support CRISPR interference. Because crRNA-effector complexes such as Cascade, Cas9, and Cas12a rely on PAMs to find their target DNA and to avoid host CRISPR arrays, they noted, the selection of PAM-compliant spacers enhances the success rate of CRISPR interference.

Other than influencing the PAM, the researchers also found that Cas4 may have a role in processing spacer substrates before or during integration. The variable spacer size is dictated by the Cas1-2 complex from the type I-D CRISPR system. "This spacer size variation is observed not only in type I-D but also in type I-B and many CRISPR systems containing cas4 genes. Our data are consistent with a model in which the nuclease activities of Cas4 tailor prespacer substrates for the Cas1-2 adaptation machinery during the integration of new spacers," the authors wrote.

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