Inducible in vivo genome editing with CRISPR-Cas9

Abstract

CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

Figure 1

Inducible genome editing in mESCs. A. Schematic representation of the c3GIC9 col1a1-targeting vector. U6-sgRNA cassettes are cloned (NsiI/SbfI) into a unique NsiI site, recreating the NsiI site upstream of the U6 promoter, allowing addition of further U6-sgRNA modules. B. GFP induction in ES cell clones 72 hrs following 1μg/ml dox treatment. C. Surveyor assay of the CR8, Apc and Trp53 loci following 2 days of dox treatment of individual targeted ES cell clones (2 clones shown per genotype). Asterisk indicates detectable mutation events in the absence of dox observed in some clones. D. Indel frequency at the Apc locus in transgenic c3GIC9-Apc/Trp53 ESCs culture with or without 1μg/ml dox as indicated (Error bars are SEM, n = 2). E. Frequency of wild-type, mono-allelic or bi-allelic indels at the Apc and Trp53 loci in c3GCI9-Apc/Trp53 ESCs treated with dox for 10 days, then expanded as individual clones in the absence of dox (clone A, n = 83; Clone B, n = 71). F. Frequency of frameshift (blue) and in-frame mutations (green) at the Apc and Trp53 loci in ESC clones described in E. G. Pie charts representing the frequency of indels (left) and frameshift mutations (right) at both Apc and Trp53, showing 91-94% of clones carry bi-allelic indels at both loci and 40-49% carry frameshift mutations in all 4 alleles.

Figure 2

Inducible genome editing in adult mice. A. Immunofluorescent stains of intestinal sections from 10-day dox-treated R26-rtTA / c3GIC9-CR8, Apc, and Apc/Trp53 mice, as indicated. Sections, stained for Apc (red), and p53 (green) show loss of each target protein following dox treatment. White arrow indicates rare clusters of ‘p53 high’ cells in dox-treated c3GCI9-Apc/Trp53 animals. Scale bars are 50μm. B. Immunohistochemical and immunofluorescent images of intestinal sections from 10-day dox-treated R26-rtTA / c3GIC9-CR8, Apc, Apc/Trp53 and c3GIC9n-Apc (Apcⁿ) mice. Sections were stained for markers of proliferation (Ki67-green), differentiation (K20 – red) and Paneth cells (Lysozyme – brown). Induction of Apc-targeted sgRNAs results in hyperplastic overgrowth, blocked differentiation and ectopic production of Paneth cells. Scale bars are 100μm C. Brightfield images of organoid cultures from R26-rtTA / c3GIC9-Apc transgenic mice treated with dox (0.5μg/ml) for 0, 2 4, or 7 days then expanded in culture for 10 days. D. Quantitation of spheroid formation in cultures in C. Bars represent the percentage of spheres (vs. normal organoids) 10 days following the withdrawal of dox. E. Western blot of whole cell lysates from c3GIC9-Apc and c3GIC9-Apc/Trp53 organoid cultures treated with dox as indicated shows loss of full-length Apc, an increase in non-phosphorylated β-catenin, reflecting elevated signaling following loss of Apc protein, and deletion of p53 protein only in c3GIC9-Apc/Trp53 cells. An uncropped, full-length image of the Apc blot is presented in Supplementary Figure 3, showing expression of the truncated protein following CRISPR-mediated editing.

Figure 3

Frequency and type of gene modifications following Cas9/Cas9n induction in mice. A. Frequency of indels in the intestine (dark blue) and thymus (light blue) in control (CR8), dox-naïve (no R26-rtTA) and D10 dox-treated mice at the Apc and Trp53 loci, as indicated (Error bars are SEM, n ≥ 3). B. Scatter plots displaying frequency and size of indels at the Apc and Trp53 loci in dox-treated intestine, as indicated. Blue points indicate frameshift mutations while green points indicate in-frame indels (n=4). C. Scatter plot showing a correlation of the frequency of Apc and Trp53 indels in c3GIC9-Apc/Trp53 tandem mice. Data from both intestine and thymus is shown together. D. Frequency of frameshift (blue) and in-frame indels (green) in the intestine and thymus of dox-treated mice, as indicated (n=4 for each locus and each tissue)