Structural Basis for Self-Cleavage Prevention by Tag:Anti-Tag Pairing Complementarity in Type VI Cas13 CRISPR Systems
We performed in vitro RNA targeting assays and in vivo RNA interference assays for Cas13a systems in presence or absence of anti-tag complementarity, as well as on key mutants. Our in vivo and in vitro data elucidate the molecular principles underlying anti-tag-mediated inhibition in the Cas13a system.
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The in vitro RNA cleavage assays were performed by mixed Cas13a, crRNA, target RNAs at cleavage buffer at 37°C and quenched at 75°C for 5 min. Samples were analyzed by 10% TBE Urea gels. In vivo RNA interference assays were performed in E.coli cells. Cas13a and CRISPR array targeting EYFP mRNA were subcloned into pRSFDuet vector, while eyfp gene was inserted into a tetracycline-inducible pBR322 vector. The two plasmids were co-transformed into E.coli cells and cultured with double selection. Further information can be found in STAR methods and fulfilled by Lead contact.