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CRIPTIC-12a: A novel CRISPR Programmable Transposon Insertion system using Cas12a (Cpf1)

O'Sullivan, Harrison (2020) CRIPTIC-12a: A novel CRISPR Programmable Transposon Insertion system using Cas12a (Cpf1). Honours thesis, Murdoch University.

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The Acidaminococcus sp. enzyme AsCpf1 has recently been adopted for gene editing purposes since it can be guided to a T-rich PAM recognition site instead of the G-rich PAM recognition site of the widely used SpCas9. Both systems engineer targeted double stranded breaks in DNA that can be repaired by non-homologous end-joining (NHEJ) or homology directed repair (HDR) mechanisms to generate genetic mutations in eukaryotes.

In prokaryotes that lack NHEJ and HDR repair processes, random mutation can be introduced via transposase enzymes that cleave dsDNA and insert transposable elements into these locations. The TnpATn5 protein, native to the prokaryote Escherichia coli, binds to a set of inverted terminal repeats that flank the transposon Tn5, or its derivatives, and excises the element before randomly inserting this transposable element into a gene to create a knock-out mutation.

In this thesis a novel strategy was designed to fuse a catalytically inactive AsdCpf1 endonuclease to TnpATn5-NB/HYP (a modified Escherichia coli Tn5 transposase) to produce the chimeric protein AsdCpf1-TnpATn5-NB/HYP that could be used to produce programmable transposition. Domain and 3D protein analysis were used to theorize the best structural configuration of the fusion between AsdCpf1 and TnpATn5-NB/HYP. The TnpATn5-NB/HYP component was engineered to contain six mutations; four of which were used to make it non-biased, and two of which were used to make the transposase hyperactive.

To ascertain whether the fused protein components were functional, two highly regulated modular bioassays were developed and implemented. A tripartite plasmid bioassay was used to detect repression by AsdCpf1 or AsdCpf1-TnpATn5-NB/HYP, and a quadripartite plasmid bioassay was used to detect non-targeted and targeted insertion events catalyzed by AsdCpf1-TnpATn5-NB/HYP.

The core plasmid system was tripartite and contained compatible oriV elements and distinct antibiotic resistance markers to force for plasmid retention. The first plasmid was engineered to carry the programmable guide production module gCPF1 and the AsdCpf1 or AsdCpf1-TnpATn5-NB/HYP effector modules. The “CHOPCHOP” algorithm predicted guides were cloned into the gCPF1 module at directional BsaI sites. The second plasmid contained the lacIq regulatory component, and either the lacZα (colourimetric detection) or sacB (viability assay) genes required for the repression bioassays. The third plasmid contained a LacIq regulated T7p07 gene, the product of which transcribed the crRNA in the gCPF1 module. This system could be switched from an OFF state to an ON state in the absence or presence of IPTG, respectively. In the ON state, the single copy lacZω sequence on the E. coli chromosome was noticeably repressed by two of the three guides complexed with AsdCpf1. The sacB sequence introduced on a multi-copy plasmid could only be noticeably repressed by one of the guides complexed with AsdCpf1. This thesis showed repression was highly dependent on both guide sequence location and gene copy number. The assay repeated with the chimeric protein revealed that repression was occurring, albeit at a diminished level.

The core AsdCpf1-TnpATn5-NB/HYP system was expanded to a quadripartite bioassay that included a suicidal R6K plasmid delivering a mTn5 transposable element. Using this system in the OFF state, transposition was shown to occur into random sites that excluded lacZω. In contrast, using this system in the ON state, transposition only occurred in the lacZω gene, revealing functional guided transposition could be obtained. The successful creation of the chimeric AsdCpf1-TnpATn5-NB/HYP protein in this thesis provides an essential foundation for further work developing Cas-Tnp fusion proteins for work in prokaryotic hosts. It also provides another step towards large-scale gene silencing and editing with extraordinary accuracy.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): College of Science, Health, Engineering and Education
Supervisor(s): Reeve, Wayne and Tiwari, Ravi
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