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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.

SUBMITTER: Lundin A

PROVIDER: S-EPMC7525522 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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

Lundin Anders A Porritt Michelle J MJ Jaiswal Himjyot H Seeliger Frank F Johansson Camilla C Bidar Abdel Wahad AW Badertscher Lukas L Wimberger Sandra S Davies Emma J EJ Hardaker Elizabeth E Martins Carla P CP James Emily E Admyre Therese T Taheri-Ghahfarokhi Amir A Bradley Jenna J Schantz Anna A Alaeimahabadi Babak B Clausen Maryam M Xu Xiufeng X Mayr Lorenz M LM Nitsch Roberto R Bohlooly-Y Mohammad M Barry Simon T ST Maresca Marcello M

Nature communications 20200929 1

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 generat ...[more]

PMID: 32994412

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Project description:BackgroundLarge-scale genetic screening using CRISPR-Cas9 technology has emerged as a powerful approach to uncover and validate gene functions. The ability to control the timing of genetic perturbation during CRISPR screens will facilitate precise dissection of dynamic and complex biological processes. Here, we report the optimization of a drug-inducible CRISPR-Cas9 system that allows high-throughput gene interrogation with a temporal control.ResultsWe designed multiple drug-inducible sgRNA expression vectors and measured their activities using an EGFP gene disruption assay in 11 human and mouse cell lines. The optimal design allows for a tight and inducible control of gene knockout in vitro, and in vivo during a seven-week-long experiment following hematopoietic reconstitution in mice. We next performed parallel genome-wide loss-of-function screens using the inducible and constitutive CRISPR-Cas9 systems. In proliferation-based dropout screens, these two approaches have similar performance in discriminating essential and nonessential genes. In a more challenging phenotypic assay that requires cytokine stimulation and cell staining, we observed similar sensitivity of the constitutive and drug-induced screening approaches in detecting known hits. Importantly, we demonstrate minimal leakiness of our inducible CRISPR screening platforms in the absence of chemical inducers in large-scale settings.ConclusionsIn this study, we have developed a drug-inducible CRISPR-Cas9 system that shows high cleavage efficiency upon induction but low background activity. Using this system, we have achieved inducible gene disruption in a wide range of cell types both in vitro and in vivo. For the first time, we present a systematic side-by-side comparison of constitutive and drug-inducible CRISPR-Cas9 platforms in large-scale functional screens. We demonstrate the tightness and efficiency of our drug-inducible CRISPR-Cas9 system in genome-wide pooled screening. Our design increases the versatility of CRISPR-based genetic screening and represents a significant upgrade on existing functional genomics toolbox.

Project description:PurposeIn the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter.ProceduresIn this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR.ResultsIn Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division.ConclusionsOverall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Dataset Information

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

Publications

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

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