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DUAL PATHWAY TRANSPOSITION WITH Tn7-LIKE ELEMENTS: SAFE SITES, MOBILE PLASMIDS, AND CRISPR-Cas

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dc.contributor.authorPetassi, Michael Thomas
dc.contributor.chairPeters, Joe E.
dc.contributor.committeeMemberChappie, Joshua S.
dc.contributor.committeeMemberAlani, Eric
dc.date.accessioned2021-03-12T17:40:02Z
dc.date.available2021-03-12T17:40:02Z
dc.date.issued2020-08
dc.description242 pages
dc.description.abstractMobile genetic elements are pervasive in all domains of life. In prokaryotes, mobile genetic elements facilitate the horizontal transmission of genetic information between diverse organisms as a driving force in evolution. Transposons are mobile genetic elements that exist as discrete DNA sequences capable of mobilizing between positions in a genome. The bacterial transposon Tn7 and related elements in the Tn7-like family are notable for the high degree of control in selection of target sites for transposition, featuring two discrete pathways to different classes of targets – the attachment site and dissemination pathways. In the attachment site pathway insertions are directed to a conserved safe haven in bacterial chromosomes, while in the dissemination pathway insertions are preferentially directed into other mobile genetic elements capable of cell-to-cell transfer (principally conjugal plasmids) to take advantage of horizontal transmission functions. The well-studied prototypic Tn7 utilizes five core genes tnsABCDE to accomplish the dual-pathway lifestyle. TnsABC form the core machinery that carries out the transposition reaction into positions selected by TnsD or TnsE. TnsD is sequence-specific DNA binding protein that directs insertions into the attachment site (attTn7) and TnsE recognizes features of conjugal replication to direct insertions into mobilizing plasmids. In this work, I present a combination of informatics and experimental evidence that indicates this dual-pathway lifestyle is maintained in the broader Tn7-like family by a diverse set of target selection modules. By analyzing the most abundant relatives of Tn7 without TnsD/E homologs, I can propose putative target site selector proteins that appear to functionally substitute for TnsD/TnsE to allow dual-pathway transposition in the Tn6230-like and Tn6022-like families. Next, I establish an adapted pipeline for high throughput transposition mapping with prototypic Tn7 to further investigate the nature of in vivo targeting by TnsE and provide a mapping procedure applicable to any transposon. Furthermore, I present exciting work with a group of Tn7-CRISPR-Cas elements that evolved a system of guide RNA categorization to achieve the dual-pathway lifestyle exclusively using RNA-guided transposition. Specialized guide RNAs allow long-term memory for access to chromosomal attachment sites upon entry into new hosts while conventional CRISPR features maintain the ability to continually acquire guide information to new plasmid and bacteriophage targets. Chromosome-targeting guide RNAs in the transposon are privatized from conventional CRISPR-Cas systems by sequence-specialization and a selective regulation scheme to avoid toxic self-targeting. I can utilize specialized guide RNAs to demonstrate up to 50% insertion frequency to programmed sites in a heterologous E. coli host as a powerful genetic modification tool.
dc.identifier.doihttps://doi.org/10.7298/n28h-2w15
dc.identifier.otherPetassi_cornellgrad_0058F_12248
dc.identifier.otherhttp://dissertations.umi.com/cornellgrad:12248
dc.identifier.urihttps://hdl.handle.net/1813/102964
dc.language.isoen
dc.subjectCRISPR-Cas
dc.subjectTn7
dc.subjectTn7-CRISPR-Cas
dc.subjectTransposition
dc.titleDUAL PATHWAY TRANSPOSITION WITH Tn7-LIKE ELEMENTS: SAFE SITES, MOBILE PLASMIDS, AND CRISPR-Cas
dc.typedissertation or thesis
dcterms.licensehttps://hdl.handle.net/1813/59810
thesis.degree.disciplineMicrobiology
thesis.degree.grantorCornell University
thesis.degree.levelDoctor of Philosophy
thesis.degree.namePh. D., Microbiology

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