CRISPR-Cas systems offer powerful and versatile biomolecular tools with many applications including genome editing, gene regulation, sequence-specific antimicrobials, and imaging. CRISPR-Cas systems rely on ribonucleoproteins as effector complexes with the CRISPR RNA driving target recognition through base-pairing interactions. Despite the critical role of the CRISPR RNA in target recognition, relatively little is known about how it can be modified. One commonly held position is that limited changes can be made to the length of the CRISPR RNA. To probe the contribution of CRISPR RNA length, we converted the <i>Escherichia coli</i> Type I-E CRISPR-Cas system into a programmable repressor through the deletion of the system’s endonuclease gene and measured silencing efficiency using a fluorescent reporter construct and flow cytometry. Here, we report that the length of Type I-E CRISPR RNAs is not fixed and can be extended while maintaining functionality. Interestingly, altering RNA length can elicit significant improvement in transcriptional silencing efficiency and specificity, but only for particular target locations. These findings heighten our understanding of CRISPR-Cas systems as they continue to increase in popularity and may offer insight into designing tunable transcriptional regulators with enhanced target specificity.

RNA length affects form and function of the Type I CRISPR-Cas surveillance complex

251st National Meeting (2016)

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