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

Anders Lundin,Carla P Martins,Maryam Clausen,Lorenz M Mayr,Camilla Johansson,Jenna Bradley,Abdel Wahad Bidar,Lukas Badertscher,Elizabeth Hardaker,Anna Schantz,Michelle J Porritt,Emily James,Xiufeng Xu,Frank Seeliger,Himjyot Jaiswal,Sandra Wimberger,Roberto Nitsch,Amir Taheri‐Ghahfarokhi ,Babak Alaei-Mahabadi ,Emma Davies ,Mohammad Bohlooly‐Y ,Therése Admyre ,Simon T Barry,Marcello Maresca

doi:10.1038/s41467-020-18548-9

Anders Lundin, Carla P Martins + Show 22 more

Open Access

https://doi.org/10.1038/s41467-020-18548-9

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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.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas[9] system has increased the speed and precision of genetic editing in cells and animals
As the ODInCas[9] system is an all-in-one system it allows for rapid cell line generation by a single transgene integration event compared to split Tet-On element systems[33]
In comparison to other inducible systems[34], the use of ObLiGaRe allowed for targeted integration of the large transgene (ODInCas[9] cassette) by zinc finger nucleases (ZFNs) homologies in both human and mouse genomic backgrounds, in parallel

Summary

Introduction

The CRISPR-Cas[9] system has increased the speed and precision of genetic editing in cells and animals. One strategy has been to exploit nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) for targeted insertions and deletions (indels) or precise genome edits[1,2,3,4] Among those nucleases, the CRISPRCas[9] system has revolutionized precision molecular genetic approaches, accelerating the generation of genetically engineered cells and animal models (reviewed in Adli[5]). Cancer prone non-germline chimeric mice, produced by injecting genetically engineered embryonic stem cells into blastocysts of a mouse line, have an increased variability related to tumorigenesis between individual mice[11] This negatively affects standardizing treatment studies in these models. The introduction of constitutive expressing Cas[9] mice, Rosa26Cas[9], reduced the number of components that needed to be delivered to the cell thereby increasing editing efficacy[13,14]

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