Inducible Genome Editing with Conditional CRISPR/Cas9 Mice

Abstract

Genetically engineered mouse models (GEMMs) are powerful tools by which to probe gene function in vivo, obtain insight into disease etiology, and identify modifiers of drug response. Increased sophistication of GEMMs has led to the design of tissue-specific and inducible models in which genes of interest are expressed or ablated in defined tissues or cellular subtypes. Here we describe the generation of a transgenic mouse harboring a doxycycline-regulated Cas9 allele for inducible genome engineering. This model provides a flexible platform for genome engineering since editing is achieved by exogenous delivery of sgRNAs and should allow for the modeling of a range of biological and pathological processes.

A Tet/Q Hybrid System for Robust and Versatile Control of Transgene Expression in C. elegans

“…Following 12 hr of Dox exposure (1 ng/μL), GFP was effectively induced throughout the body, although GFP intensities were rather punctate instead of uniform across various regions (Figure 2B, left, GFP ). As rtTA expression level can dictate TRE activity (Katigbak et al., 2018, McJunkin et al., 2011), the variation in GFP expression might be caused by variable rtTA expression and thus reflected the property of Prpl-28 rather than an inherent limitation of the hybrid system. This seems the case, because the GFP signals in general coincided with the mKate signals marking rtTA expression (Figure 2B, left, Overlay ), with exceptions sometimes observed at the first pharyngeal bulb and tail tip (asterisks, Figure 2B).…”

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

“…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 18,19,35,36 . Tight regulation of Cas9 expression in the ODInCas9 system overcomes the leaky nature of Cre or Cas9 reported in other models 18 .…”

“…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] .…”

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

“…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].…”