Generating viable mice with heritable embryonically lethal mutations using the CRISPR-Cas9 system in two-cell embryos

Abstract

A substantial number of mouse genes, about 25%, are embryonically lethal when knocked out. Using current genetic tools, such as the CRISPR-Cas9 system, it is difficult-or even impossible-to produce viable mice with heritable embryonically lethal mutations. Here, we establish a one-step method for microinjection of CRISPR reagents into one blastomere of two-cell embryos to generate viable chimeric founder mice with a heritable embryonically lethal mutation, of either Virma or Dpm1. By examining founder mice, we identify a phenotype and role of Virma in regulating kidney metabolism in adult mice. Additionally, we generate knockout mice with a heritable postnatally lethal mutation, of either Slc17a5 or Ctla-4, and study its function in vivo. This one-step method provides a convenient system that rapidly generates knockout mice possessing lethal phenotypes. This allows relatively easy in vivo study of the associated genes' functions.

Fig. 1

Generation of heritable founder (F0) mice with Virma or Dpm1 insertion/deletion mutations. a Schematic diagram of sgRNA targeting sites in the mouse Virma locus. PAM = protospacer adjacent motif. b Sequencing traces of PCR products encompassing the Virma target region from wild-type (wt) mice and from representative mutant F0 mice (#9 and #10), generated by microinjecting Cas9 mRNA and sgRNA1 or sgRNA2 into one blastomere of two-cell embryos, and their filial generation (F1). Overlapping sequencing traces among the mutant mice indicate the presence of more than one allele among these mice, compared with wt mice. The mutations’ start positions are indicated by black arrows. c PCR sequences from representative mutant mice generated by microinjecting different sgRNA targeting sites into one blastomere of two-cell stage embryos. PCR products were analyzed with Sanger sequencing. Deleted nucleotides are indicated by red hyphens. d Schematic diagram of sgRNA targeting site in the mouse Dpm1 locus. e Sequencing traces of PCR products encompassing the Dpm1 target region from wt mice and from a representative mutant F0 mouse and one of its offspring (F1). Overlapping sequencing traces among the mutant mice indicate the presence of more than one allele among these mice, compared with wt mice. The mutations’ start positions are indicated by black arrows. f PCR sequences from wt and a representative mutant mouse produced by microinjection into one blastomere of a two-cell stage embryo. PCR products were analyzed with Sanger sequencing. The mutated base is labeled in red

Fig. 2

Heritable F0 mice with Slc17a5 or Ctla-4 insertion/deletion mutations. a Schematic diagram of the sgRNA targeting site in the mouse Slc17a5 locus. PAM = protospacer adjacent motif. b PCR sequences encompassing the Slc17a5 target region from wild-type (wt) mice and from representative mutant mice (#4 and #16) generated by two methods of microinjection: (1) into zygotes using sgRNA; and (2) into one blastomere of two-cell stage embryos using sgRNA. PCR products were sequenced and those showing overlapping sequencing traces were cloned. The subsequent individual clones were then sequenced. Mutated bases are labeled in red and deleted nucleotides are indicated by hyphens. Numbers and percentages of the mutated bases are listed on the right. c Sequencing traces of PCR products from representative mutant mice generated by the aforementioned two microinjection methods. Overlapping sequencing traces among the mutant mice indicate the presence of more than one allele among these mice, compared with wt mice. The mutations’ start positions are indicated by black arrows. d Germline transmission of the Slc17a5 lethal mutations in F1 progeny is shown by sequencing traces of PCR products encompassing the Slc17a5 target region from a representative mutant F1 mouse. Overlapping sequencing traces indicate heterozygous mice. The black arrow shows the mutation starting position. e Schematic diagram of the sgRNA targeting site in the mouse Ctla-4 locus. f Sequencing traces of PCR products encompassing the Ctla-4 target region from wt mice and from a representative mutant F0 mouse. The mutant mouse’s overlapping sequencing traces indicate the presence of more than one allele, compared with wt mice. The mutation start position is indicated by a black arrow. g PCR sequences of the Ctla-4 target region of wt mice and a representative mutant mouse generated by microinjection into one blastomere of two-cell embryos. The mutated base is labeled in red and deleted nucleotides are indicated by hyphens

Fig. 3

Off-targets identified from Slc17a5 F0 mice. Sequencing traces of PCR products encompassing the target regions of Virma a, Ctla-4 b, or Slc17a5 c in the tissues or Treg cells of representative mutant mice produced by the OSTCM method. Overlapping sequencing traces indicate the presence of more than one allele among the mutant mice. Editing (indel) efficiencies assessed by TIDE analysis in each tissue is shown to the right of each trace. d Venn diagram displaying the overlap of all single-nucleotide variants detected in whole-genome sequencing data from three mutant mice (#10, #17, and #18). e The top ten potential off-target sites of CRISPR-Cas9 for sgRNA. Red nucleotides represent a mismatch compared with the on-target sequences. Ten selected potential off-target sites analyzed with Sanger sequencing of the livers of the three mutant mice from d

Fig. 4

Virma knockout led to developmental glomerulus abnormalities in adult mouse kidneys. a Two images, top left: in situ hybridization (ISH) of Virma mRNA in the kidneys of wild-type (wt) mice and of Virma knockout F0 mice generated by the OSTCM method, scale bar: 100 µm; bar chart, top right: quantification of mRNA; two images, lower left: hematoxylin/erythrosine (HE) stained kidney glomeruli from wt mice and from Virma knockout F0 mice, scale bar: 100 μm; lower right: glomerulus diameters. b q-PCR results of the differential expression of Virma mRNA in kidney tissue between wt mice and knockout F0 mice. c RT-PCR products of Virma mRNA from the kidney tissue of knockout F0 mice were cloned and sequenced. Upper panel: mutated bases are labeled in red and deleted nucleotides are indicated by hyphens. Numbers and percentages of the mutated bases are listed on the right. Lower panel: sequencing traces of RT-PCR products. The mutant mouse’s overlapping sequence traces indicate the presence of more than one allele. The mutation start position is indicated by a black arrow. d, e Confocal microscopy of C3 (d) or IgM (e) deposits in the kidney glomeruli in adult Virma knockout F0 mice. DAPI (blue), C3 protein or IgM protein (green). Scale bar: 50 μm. f Differentially expressed genes (fold change > 1.5 or < 0.5) identified by RNA-seq in kidney tissue from both wt mice and Virma mutant mice. g Gene ontology (GO) and enrichment analysis of the differentially expressed genes. Values are means ± SEM. Asterisks in all plots, P-values after unpaired t test, two-tailed, * indicates significance at P < 0.05, ** indicates significance at P < 0.01. Source data are provided as a Source Data file

Fig. 5

Slc17a5 knockout diminished sialic acid and nitrate secretory functions. a In situ hybridization (ISH) and immunohistochemistry (IHC) results for the presence of Slc17a5 mRNA and sialin protein in tissue slices of submandibular glands of wt mice and Slc17a5 knockout F0 mice generated by the OSTCM method. Quantifications of mRNA and protein are shown in panels on the right. Scale bar: 50 µm. b q-PCR results show differing expressions of Slc17a5 mRNA in submandibular glands between wt mice and Slc17a5 knockout F0 mice. c IHC (left panels) and quantification (right panels) of transmembrane protein 16 A (TMEM16A) and aquaporin 5 protein (AQP5) in submandibular glands of wt mice and of Slc17a5 knockout F0 mice. Scale bar: 50 µm. d Comparison of total sialic acid in saliva, after water-immersion-restraint stress of wt mice and Slc17a5 knockout F0 mice. e Comparison of NO₃⁻ in saliva and serum after water-immersion-restraint stress of both wt mice and Slc17a5 knockout F0 mice. Values are means ± SEM. Asterisks in all plots, P-values after unpaired t test, two-tailed, * indicates significance at P < 0.05, ** indicates significance at P < 0.01. Source data are provided as a Source Data file

Fig. 6

Activation of Treg cells by Ctla-4 knockout in adulthood. a Frequencies of CD25(+)CD127(−) (Up) and Foxp3(+) (Down) Treg cells in spleens of Ctla-4 knockout and wt mice. Representative fluorescence-activated cell sorting (FACS) results are in the left panels, and the corresponding statistical results are in the right panel. n = 4–5 mice per group. b Single-cell genotyping of a single CD3(+)CD4(+)CD25(+)CD127(−) Treg cell from the spleen of one mutant chimera. The cell was PCR amplified and Sanger sequenced. Sequencing traces of PCR products from representative wt and mutant cells generated by the OSTCM method are shown. The mutations’ start positions are indicated by black arrows. Numbers and percentages of the mutated cells are listed on the right. c, d Frequencies of CTLA-4(+) cells among CD3(+)CD4(+)CD25(+)CD127(−) Treg cells in the spleens of Ctla-4 knockout and wt mice before (c) and after (d) in vitro treatment (aCD3: 3 μg/mL, aCD28: 2 μg/mL, and IL2: 20 ng/mL for 3 days). Representative FACS results are in the left panels, and the corresponding statistical results are in the right panels. n = 3–5 mice per group. Values are means ± SEM. Asterisks in all plots, P-values after unpaired t test, two-tailed, * indicates significance at P < 0.05, ** indicates significance at P < 0.01. Source data are provided as a Source Data file

Fig. 7

Generation of surviving chimeric mice with lethal mutations. Compared with zygote microinjection, microinjecting CRISPR-Cas9 components into one blastomere of two-cell embryos can generate surviving adult chimeric mice with lethal mutations