A Convenient Cas9-based Conditional Knockout Strategy for Simultaneously Targeting Multiple Genes in Mouse

Abstract

The most powerful way to probe protein function is to characterize the consequence of its deletion. Compared to conventional gene knockout (KO), conditional knockout (cKO) provides an advanced gene targeting strategy with which gene deletion can be performed in a spatially and temporally restricted manner. However, for most species that are amphiploid, the widely used Cre-flox conditional KO (cKO) system would need targeting loci in both alleles to be loxP flanked, which in practice, requires time and labor consuming breeding. This is considerably significant when one is dealing with multiple genes. CRISPR/Cas9 genome modulation system is advantaged in its capability in targeting multiple sites simultaneously. Here we propose a strategy that could achieve conditional KO of multiple genes in mouse with Cre recombinase dependent Cas9 expression. By transgenic construction of loxP-stop-loxP (LSL) controlled Cas9 (LSL-Cas9) together with sgRNAs targeting EGFP, we showed that the fluorescence molecule could be eliminated in a Cre-dependent manner. We further verified the efficacy of this novel strategy to target multiple sites by deleting c-Maf and MafB simultaneously in macrophages specifically. Compared to the traditional Cre-flox cKO strategy, this sgRNAs-LSL-Cas9 cKO system is simpler and faster, and would make conditional manipulation of multiple genes feasible.

Figure 1

Generation of transgenic Cas9-based EGFP conditional KO mouse. (a) Schematic of sgRNAs^EGFP-UbC-LSL-Cas9 transgenic vector. Four sgRNAs (pink) driven by U6 promoters (green arrowheads) were used to target independent sites of EGFP coding sequence. The Cas9 with nuclear localization signal (NLS) and FLAG tag was fused to LSL element and driven by UbC promoter. (b) DsRed fluorescence in organs of sgRNAs ^EGFP-LSL-Cas9 mouse (Tg) line 27. The upper panel shows organs in bright field. The lower panel shows dsRed fluorescence. Dotted lines indicate the organs invisible. Scale bar, 5 mm. (c) DsRed mRNA expression level in organs of mouse line 27 was analyzed by RT-PCR. The expression level of dsRed relative to β-actin in different organs was normalized to that in brain. WT mouse RNA mixture was absent of dsRed mRNA and used as a negative control (n = 3 repeats).

Figure 2

Tissue-specific mutations of EGFP in sgRNAs ^EGFP-LSL-Cas9; EGFP; Alb-Cre mice. (a,b) EGFP fluorescence of liver was almost completely eliminated in sgRNAs ^EGFP-LSL-Cas9; EGFP; Alb-Cre (Alb-Cre) mouse. Control (Ctrl) was sgRNAs ^EGFP-LSL-Cas9; EGFP mouse. The right panels quantified fluorescence. (a) Paired t-test. liver, *p = 0.0109; brain, p = 0.8084, not significant (ns); heart, p = 0.8893, ns; kidney, p = 0.3101, ns. n = 3. (b) Paired t-test, liver, ***p = 0.0002; brain, p = 0.1486, ns; heart, p = 0.8784, ns; kidney, p = 0.2086, ns. n = 3. Scale bar, 5 mm for (a); 200 μm for (b). (c) T7EN1 assay showed obvious digested bands (arrows) indicating mutations in the lane of Alb-Cre positive mouse, while none in Cre negative mouse (Ctrl). M, maker. (d) Mutated EGFP sequences from sgRNAs ^EGFP-LSL-Cas9; EGFP; Alb-Cre mouse liver. PAM (Red), sgRNAs targeting sites (yellow grounding), deletion (grey grounding) and base changing (blue grounding) are shown. Δ, deletion; m, mutation; ×, no mutation in the presenting sequence. Numbers following the symbols are base quantity of mutation.

Figure 3

c-Maf/MafB double cKO via sgRNAs-LSL-Cas9 system. (a) Schematic of transgenic vector sgRNAs^c-Maf/MafB-UbC-LSL-Cas9. Three U6 promoters (green arrowheads) driven c-Maf sgRNAs (blue blocks) and three U6 promoters driven MafB sgRNAs (green blocks) were arranged alternately. Other functional regions are combined as those in the sgRNAs^EGFP-UbC-LSL-Cas9 transgenic vector (Fig. 1a). (b) Photograph of sgRNAs ^c-Maf/MafB-UbC-LSL-Cas9 mice. Ctrl, transgenic mouse without Cre. LysM-Cre, transgenic mouse express lysM-Cre. (c) Western blots of c-Maf/MafB double cKO macrophages (paired t-test, c-Maf, ***p = 0.003, n = 3. MafB, ***p = 0.0009, n = 3). (d) T7EN1 analysis of c-Maf and MafB mutations in the macrophages of a sgRNAs ^c-Maf/MafB-LSL-Cas9; LysM-Cre (LysM-Cre) mouse. The control (Ctrl) was a sgRNAs ^c-Maf/MafB-LSL-Cas9 mouse without Cre recombinase. M, marker. (e) c-Maf mutation mode of the Cre-expressing macrophages of the transgenic mouse. (f) MafB mutation mode of the Cre-expressing macrophages of the transgenic mouse.+, insertion.