Highly efficient gene knockout in mice and zebrafish with RNA-guided endonucleases

Abstract

RNA-guided endonucleases (RGENs), derived from the prokaryotic Type II CRISPR-Cas system, enable targeted genome modification in cells and organisms. Here we describe the establishment of gene-knockout mice and zebrafish by the injection of RGENs as Cas9 protein:guide RNA complexes or Cas9 mRNA plus guide RNA into one-cell-stage embryos of both species. RGENs efficiently generated germline transmittable mutations in up to 93% of newborn mice with minimal toxicity. RGEN-induced mutations in the mouse Prkdc gene that encodes an enzyme critical for DNA double-strand break repair resulted in immunodeficiency both in F₀ and F₁ mice. We propose that RGEN-mediated mutagenesis in animals will greatly expedite the creation of genetically engineered model organisms, accelerating functional genomic research.

Figure 1.

Generation of a RGEN specific for the Prkdc gene. (A) A schematic diagram depicting a target-specific single guide RNA (sgRNA). (B) RGEN target sites in exon 2 of the mouse Prkdc gene. PAMs are shown in red and targets are denoted by black lines. (C) In vitro cleavage assays evaluating Prkdc-RGEN activity. The arrow indicates bands cleaved by RGENs.

Figure 2.

RGEN-induced Prkdc gene disruption in mice. (A) Representative T7E1 assays demonstrating mutagenesis efficiencies of Cas9 mRNA plus Prkdc-specific sgRNA that were delivered via intracytoplasmic injection into one-cell-stage mouse embryos. Numbers indicate independent founder mice generated from the highest dose. Arrows indicate bands cleaved by T7E1. fPCR results (B) and DNA sequences of mutant alleles (C) observed in three Prkdc mutant founders denoted in red in A. The red arrowhead in C indicates the cleavage site by the RGEN.

Figure 3.

RNA-guided mutagenesis in zebrafish embryos. (A,B) Target sequences (upper) and RGEN-induced indels detected by T7E1 assays (lower). The Cas9 protein–sgRNA complex was microinjected into one-cell-stage embryos with concentrations of 1 ng, 4 ng, or 8 ng per embryo. (C) Time courses of Cas9 protein:sgRNA and Cas9 mRNA:sgRNA injection into embryos during early development. Arrows indicate bands cleaved by T7E1. (hpi) Hours post-injection.

Figure 4.

Analysis of off-target activity of RGENs in Prkdc-mutant founders. T7E1 assays were conducted using genomic DNA samples from the indicated Prkdc mutant founders. The Prkdc gene and putative off-target sites bearing 1 to 3-bp mismatches that were selected with Bowtie 0.12.9 (Langmead et al. 2009) were examined. Bi-allelic mutants containing large insertions are shown in blue (Supplemental Table 2), mismatched bases in red, and PAM in green; black lines indicate putative 11-bp seed regions.

Figure 5.

Germline transmission and mutant phenotypes of the Prkdc mutant mouse. (A) Detailed genotypes of F₀ mutants (♂ 25 and ♀ 33, upper panel) and determination of genotypes of their F₁ progenies (#1 and #7) by fPCR (lower panel). Note that a mutant allele (+2) of female founder #33 was only detected by fPCR (Supplemental Table 2). Using lymphocytes isolated from peripheral blood (PBMC), lymph node (LN), spleen, and thymus, frequencies of CD4⁺ and CD8⁺ T cells (B) and those of CD3⁺ T cells and CD19⁺ B cells (C) were determined. The abnormal frequencies of immune cells are denoted in red. BALB/c and scid mice, positive and negative controls for B and T lymphocytes, respectively.