Development of an ObLiGaRe Doxycycline Inducible Cas9 system for pre-clinical cancer drug discovery

Abstract

The CRISPR-Cas9 system has increased the speed and precision of genetic editing in cells and animals. However, model generation for drug development is still expensive and time-consuming, demanding more target flexibility and faster turnaround times with high reproducibility. The generation of a tightly controlled ObLiGaRe doxycycline inducible SpCas9 (ODInCas9) transgene and its use in targeted ObLiGaRe results in functional integration into both human and mouse cells culminating in the generation of the ODInCas9 mouse. Genomic editing can be performed in cells of various tissue origins without any detectable gene editing in the absence of doxycycline. Somatic in vivo editing can model non-small cell lung cancer (NSCLC) adenocarcinomas, enabling treatment studies to validate the efficacy of candidate drugs. The ODInCas9 mouse allows robust and tunable genome editing granting flexibility, speed and uniformity at less cost, leading to high throughput and practical preclinical in vivo therapeutic testing.

Universal toxin-based selection for precise genome engineering in human cells

“…At termination, liver tissue was collected for genomic DNA extraction and analysis by NGS. Icons of mice and syringes were created in a previous publication 84 , and were used without modification under Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/ ). b Enrichment of CBE editing at the HBEGF locus.…”

Rapid target validation in a Cas9-inducible hiPSC derived kidney model

“…In this study, we selected a doxycycline-inducible Cas9 expressing hiPSC line 21 to conduct gene-editing and differentiation experiments. The reason behind selecting the Cas9-inducible cell line in our study was to try and achieve as high a rate of editing as possible, as each cell would express Cas9 upon doxycycline induction and so only guides would need to be introduced to the cells to enable efficient editing, simplifying the transfection step.…”

A Perspective on Synthetic Biology in Drug Discovery and Development—Current Impact and Future Opportunities

“…PRIME editors, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA) offer a bespoke search-and-replace system for specific insertion, deletions and base-to-base swaps, with druginducible Cas9 allowing temporal control of gene editing for in vitro and in vivo applications at any desired point in the genome. 119 Disruptive Science CRISPR-Cas9 95,96 is revolutionizing the way we do everything, from target validation to animal model development to the way we think about new therapeutics. It is not a surprise that the 2020 Nobel Prize for Chemistry was awarded to Emmanuelle Charpentier and Jennifer Doudna for the discovery of CRISPR-Cas9 genetic scissors.…”

“…PRIME editors, consisting of a catalytically impaired Cas9 endonuclease fused to an engineered reverse transcriptase enzyme, and a prime editing guide RNA (pegRNA) offer a bespoke search-and-replace system for specific insertion, deletions and base-to-base swaps, with drug-inducible Cas9 allowing temporal control of gene editing for in vitro and in vivo applications at any desired point in the genome. 119…”