New England Southern Engineering a Termination Readthrough-Based Gene Switch Enables Controllable CRISPR Gene Editing

Overview

Gene switches that can artificially regulate gene expression are in high demand for developing safe and effective gene therapies. Using in vitro cell culture, DNA transfection, and fluorescence microscopy, I first examined efficiencies of the three stop codons in terminating protein translation. Interestingly, I found that all three stop codons were “leaky” albeit at low levels and could be subjec t to termination readthrough. I identified the stop codon UGA (or TGA in DNA), when flanked with six virus -derived downstream nucleotides, was particularly amenable to chemical modulation in its readthrough propensity. Based on these findings, I constructed a new two-component gene switch that consisted of a nine-nucleotide, TGA- comprised DNA effector and a chemical inducer. The DNA effector was designed to be inserted into the target gene of interest and to limit gene expression using the native function o f the stop codon TGA. The chemical inducer, such as the clinically available gentamicin, would be applied separately to increase readthrough of TGA thereby “switching on” target gene expression. I validated the design of such gene switch by observing its function in controlling Cre recombination in vitro. Importantly, I further demonstrated the feasibility of using this gene switch to engineer “switchable” CRISPR-Cas9 gene editing machinery that could have potential for clinical use. The extremely small size of the DNA effector, low risk of immunogenicity, and clinical track record of the chemical inducer make the gene switch I reported herein a potentially versatile tool for developing inducible cell and gene therapies.

Competition history

• JSHS 2024 Category not listed

Resources

• National JSHS abstract book ↗

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