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A novel targeted DNA insertion system using Cpf1 and Himar transposase

Rasheed, Bilal (2022) A novel targeted DNA insertion system using Cpf1 and Himar transposase. Honours thesis, Murdoch University.

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Abstract

CRISPR-Cas proteins have been exploited in genome recombineering research since they can induce targeted Double Stranded Breaks (DSBs) in the DNA. This has been particularly useful since cellular repair mechanisms will resect DSBs and introduce mutations in the process. Each Cas protein has a distinct PAM recognition site which expands the range of possible DNA targets in a genome. Catalytically dead versions of CRISPR-Cas enzymes which cannot introduce a DSB but are still able to bind to DNA has facilitated studies in gene repression and regulation. A dead-Cas9 protein has been fused to the Hsmar transposase showing a proof of concept for targeted DNA insertion.

This thesis has developed novel strategies to fuse a catalytically dead dCpf1 protein to the Mariner Himar transposase producing a protein that is able to target T-rich PAM sites for targeted DNA insertion. A strategy was designed to facilitate the cloning of the fusion under the control of the inducible promoter pTet in a p15A chloramphenicol resistant backbone. In this strategy, the final plasmid carrying this fusion was designated pDHD. This construct was designed so that different guides could easily be directionally cloned into the backbone and be transcriptionally activated by T7 RNA polymerase. This feature is particularly desirable as it allows the plasmid to target many different DNA sites.

To monitor for integration into DNA a bioassay was developed. The design included the use of Escherichia coli DH5α (pDHD, pSW213 and pT7T7) grown in the presence of anhydrous tetracycline (aTc) to induce the dCpf1-Himar fusion and IPTG to induce RNA polymerase, the guide RNA for dCpf1 and lacZα. This would enable the chromosomal lacZω to be targeted by dCpf1-Himar fusion complexed to its guide to enable incorporation of the mini-Himar transposon carrying neomycin resistance at this location. This mini-Himar transposon is located within the backbone of pSAM_Rl. Since this plasmid carried an active TnpA, this gene had to be excised by restriction digestion and religated. This gene was excised in this study and the final construct was designated pSAM_RlΔTnpA. Since this plasmid contains an R6K oriV, it would need to be electroporated into E. coli DH5α (pDHD, pSW213 and pT7T7) in order to be replicated. Transformants containing mini-Himar insertions would need to be selected as neomycin resistant white colonies on plates containing neomycin, X-Gal, aTc and IPTG.

All components were prepared for the assay except for pDHD which did not contain the Cas-TnpA fusion as expected based on the restriction profile. Further work is required to ensure the correct restriction profile is established prior to performing the bioassay. An alternative strategy to produce the Cas-TnpA fusion was designed, but due to time constraints, further work is necessary to produce the final construct. Once the final Cas-TnpA construct has been developed, successful targeted transposition could be tested using the bioassay outlined in this thesis.

The development of an inducible dCpf1-Himar transposase fusion would expand the current toolset available to scientists to perform targeted DNA insertions.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): Environmental and Conservation Sciences
Supervisor(s): Reeve, Wayne, Tiwari, Ravi and Watson, Mark
URI: http://researchrepository.murdoch.edu.au/id/eprint/66118
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