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Oral: The Function and Mechanism of CRISPR and Its Applications


Activity and repurposing of native CRISPR-CAS systems
R. BARRANGOU (1) (1) NC State University, U.S.A.

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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins collectively constitute a DNA-encoded, RNA-guided, nucleic acid-targeting adaptive immune system. Although a plethora of diverse CRISPR-Cas systems are widespread in many bacterial phyla, only a few model systems have been characterized to date. Here, we investigate the occurrence and diversity of CRISPR-Cas systems in lactobacilli and characterize their activity in native hosts. Specifically, we show that endogenous Type II systems are natively active in several Lactobacillus species and provide interference against the uptake of plasmids that carry sequences matching recently acquired spacers. We also characterize a diversity of CRISPR transcripts (crRNAs, tracrRNAs) and PAMs, and show a diversity of guide sequences and structures. Noteworthy, diverse PAMs with varying GC content provide flexibility for targeting of nucleic acid sequences in genome editing applications. We also show that some of these systems can be re-purposed as potent antimicrobials by providing self-targeting guides that co-opt the natural CRISPR machinery. Furthermore, we illustrate how self-targeting can also be used as a screening method to identify essential genes and genomic regions. Collectively, these results open new avenues for applications of Type II CRISPR-Cas systems and highlight the need to investigate and characterize CRISPR-Cas systems in native hosts.