Platforms of in vivo genome editing with inducible Cas9 for advanced cancer modeling

Abstract

The emergence of clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 technology has dramatically advanced how we manipulate the genome. Regarding in vivo experiments, Cas9‐transgenic animals could provide efficient and complex genome editing. However, this potential has not been fully realized partly due to a lack of convenient platforms and limited examples of successful disease modeling. Here, we devised two doxycycline (Dox)‐inducible Cas9 platforms that efficiently enable conditional genome editing at multiple loci in vitro and in vivo. In these platforms, we took advantage of a site‐specific multi‐segment cloning strategy for rapid and easy integration of multiple single guide (sg)RNAs. We found that a platform containing rtTA at the Rosa26 locus and TRE‐Cas9 together with multiple sgRNAs at the Col1a1 locus showed higher efficiency of inducible insertions and deletions (indels) with minimal leaky editing. Using this platform, we succeeded to model Wilms’ tumor and the progression of intestinal adenomas with multiple mutations including an activating mutation with a large genomic deletion. Collectively, the established platform should make complicated disease modeling in the mouse easily attainable, extending the range of in vivo experiments in various biological fields including cancer research.

ObLiGaRe doxycycline Inducible (ODIn) Cas9 system driving pre-clinical drug discovery, from design to cancer treatment

“…The mRNA for each of the point mutations ( Kras G12D and Kras G12C) was expressed in approximately 50% of cells within an adenocarcinoma carrying at least 1 copy of the gene. The difference in the indolence of each of the models, Kras G12D vs G12C and the Trp53 with Stk11 reveals a heterogeneity that has been reported by others [13, 17, 20] and likely results from differential usage of CRISPR-Cas9 double strand break repair mechanisms, homology directed repair, or non-homologous end joining, followed by different selective pressures over the heterogenous population of genetic mutations that arise in each model. This heterogeneity is unlike the results reported for other GEMMs and is an advantage of the inducible Cas9 systems [38].…”

“…Moreover, as the system turns off upon withdrawal of dox it can be repeatedly activated for sequential editing of multiple genes, but more importantly inactivation minimizes unwanted downstream effects of the system and Cas9 exposure enabling purer efficacy studies. This emphasizes the advantage of the ODInCas9 inducible model over Cre activated Cas9 inducible systems that are constitutively active upon recombination [17, 18, 32, 33]. Tight regulation of Cas9 expression in the ODInCas9 system overcomes the leaky nature of Cre or Cas9 reported in other models [17].…”

“…This emphasizes the advantage of the ODInCas9 inducible model over Cre activated Cas9 inducible systems that are constitutively active upon recombination [17, 18, 32, 33]. Tight regulation of Cas9 expression in the ODInCas9 system overcomes the leaky nature of Cre or Cas9 reported in other models [17]. Further, the Cre-loxP system is limited by the presence of cryptic loxP sites in the mammalian genome, thus genotoxicity is induced by Cre recombinase [21, 34] which is not observed with the doxycycline Tet-inducible systems…”

Inducible knock-out of BCL6 in lymphoma cells results in tumor stasis

“…Previous efforts to employ a DOX-inducible Cas9 approach for the conditional deletion of MCL-1 were hampered by inefficient induction of Cas9 expression [33]. In recent studies, robust expression of Cas9 in an in vivo Cas9 expressing mouse model was reported [34,35]. Here, we have demonstrated that our system permits effective, conditional expression of Cas9 in DLBCL cell lines.…”

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

“…Inducible expression systems including recombinase-mediated cassette exchange, Cre-recombinase, or tetracycline (Tet) sensitive systems, have been used to generate inducible Cas9 mice 12,13,[16][17][18][19][20][21] .…”