CRISPR-Cas defense systems are employed by their hosts to prevent parasitization by mobile genetic elements. The discovery of nuclease-deficient CRISPR-Cas systems contained within transposon ends suggested a repurposing of the contained defense system. One such Type I-F3 CRISPR-Cas system was found inside Tn6677, a Tn7-like transposon within the genome of a Vibrio cholerae strain. Tn6677 requires coordination between the contained CRISPR-Cas system and the transposition proteins for effective transposition. Isolation of this system, and reduction to its minimal components, enabled RNA-guided integration of donor DNA in Escherichia coli. Base-pairing interactions between the user-specified CRISPR RNA and the target sequence precede the integration of donor DNA approximately 49-bp downstream of the end of the target sequence. This system is specific regardless of the supplied RNA guide, and successfully integrates donors of different lengths. The donor DNA is indicated by flanking cognate transposon end sequences. While clearly functional, the mechanism by which the transposition proteins and the CRISPR-Cas proteins interact remained unclear. To this end we purified the multi-protein RNA-guided DNA binding complex (Cascade) from the transposon-encoded minimal I-F3 CRISPR-Cas system in complex with the transposition protein TniQ. De novo modeling revealed the unexpected dimerization of TniQ, and its location within the complex, bound to the Cas6-end of the transposon-encoded Type I-F3 Cascade. Additional models obtained from DNA-bound structures of the complex demonstrate initial steps in target binding alongside novel conformations of Cascade subunits. This work reveals the mechanism by which the Tn6677 components guide integration and will enable rational engineering of these systems for further experimentation and tool development.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/d8-k048-fn54 |
| Date | January 2021 |
| Creators | Halpin-Healy, Tyler Sheehan |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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