Maternal Supply of Cas9 to Zygotes Facilitates the Efficient Generation of Site-Specific Mutant Mouse Models

Abstract

Genome manipulation in the mouse via microinjection of CRISPR/Cas9 site-specific nucleases has allowed the production time for genetically modified mouse models to be significantly reduced. Successful genome manipulation in the mouse has already been reported using Cas9 supplied by microinjection of a DNA construct, in vitro transcribed mRNA and recombinant protein. Recently the use of transgenic strains of mice overexpressing Cas9 has been shown to facilitate site-specific mutagenesis via maternal supply to zygotes and this route may provide an alternative to exogenous supply. We have investigated the feasibility of supplying Cas9 genetically in more detail and for this purpose we report the generation of a transgenic mice which overexpress Cas9 ubiquitously, via a CAG-Cas9 transgene targeted to the Gt(ROSA26)Sor locus. We show that zygotes prepared from female mice harbouring this transgene are sufficiently loaded with maternally contributed Cas9 for efficient production of embryos and mice harbouring indel, genomic deletion and knock-in alleles by microinjection of guide RNAs and templates alone. We compare the mutagenesis rates and efficacy of mutagenesis using this genetic supply with exogenous Cas9 supply by either mRNA or protein microinjection. In general, we report increased generation rates of knock-in alleles and show that the levels of mutagenesis at certain genome target sites are significantly higher and more consistent when Cas9 is supplied genetically relative to exogenous supply.

Fig 1. Generation and validation of Cas9 overexpressing mice.

A) PhiC31 integrase mediated cassette exchange allows the transgenic targeting of a CAG-NLS-Cas9 overexpression cassette into the ROSA26 locus (Gt(ROSA26)Sor) in embryonic stem cells. B) Primary embryonic fibroblast cultures grown from Cas9 expressing (Cas9 +ve) or wild-type littermate embryos (Cas9 -ve) and transfected with sgRNAs targeted to exon 1 of the tumor suppressor genes Trp53 and Cdkn2a. Clusters of cell foci with an immortalized appearance are only evident in Cas9 expressing cultures, consistent with a loss-of-function of these two genes. C) Clonogenic assay of Cas9 expressing and wild-type embryonic fibroblast cultures following transfection with guide-RNAs targeted to Trp53 and Cdkn2a. Clones of cells are only seen in Cas9 expressing cultures, indicating an immortalized phenotype consistent with loss-of-function of these two genes. D) Sanger sequence traces from amplicons generated from pooled cells at the Trp53 and Cdkn2a loci in two independent Cas9 expressing fibroblast cultures transfected with sgRNAs targeted to Trp53 and Cdkn2a. The appearance of mixed sequences traces appearing near the protospacer-adjacent-motif (blue bar) are indicative of multiple indel mutations at these target sites.

Fig 2. Maternal supply of Cas9 within fertilized zygotes is sufficient for efficient genome editing.

A) Zygotes are obtained from matings between C57BL/6J stud males and heterozygous female mice, constitutively overexpressing Cas9. Microinjection of the resulting pronuclear stage zygotes was performed using sgRNA alone. B) Top panel shows target site amplification in blastocysts after microinjection of fertilized zygotes generated from heterozygous Cas9 expressing females with sgRNA against target chr17(+):34030557–34030576. Doublets, indicated by asterisks, are indicative of indel mutagenesis. Bottom panel shows the same blastocysts genotyped for the presence of the Cas9 transgene and a control gene. Genetically wild-type embryos (6 and 7) show clear mutagenesis (white arrows), proving that maternally contributed Cas9 is sufficient for genome editing.

Fig 3. Comparison of gene editing with maternal supply of Cas9 with Cas9 mRNA or protein injection.

Data in panels A, C, E, G from microinjection of sgRNA chr17(+):34030557–34030576 and panels B, D, F, H for sgRNA chr5(-):123582755-123582774.A) and B) Percentage mutation rates (mutant embryos per total number) for maternal Cas9 supply (homozygous donor and/or heterozygous donor) and exogenous Cas9 supply as mRNA or protein (wild-type donor) supply, showing similar efficiencies. C) and D) Box plot showing the range of mutagenesis (amount of wild-type sequence mutation within a single mutant blastocyst) for maternal Cas9 supply and exogenous Cas9 supply as mRNA and protein (wild-type donor). The ends of the whiskers are at 1.5 interquartile ranges above/below the third/first quartiles. E) and F) As for C) and D) but comparing mutagenesis efficiency in either heterozygous (Cas9/+) or wild-type (WT) embryos derived from microinjected zygotes obtained from heterozygous Cas9 expressing females. E) and F) Box blot showing the range of alleles identified in mutant embryos generated from maternal Cas9 supply and exogenous supply as mRNA and protein. Data inside the bars are the number of embryos sampled.

Fig 4. Comparison of maternal supply of Cas9 with exogenous Cas9 mRNA injection for the production of knock-in alleles.

A) Percentage mutation rates (mutant embryos per total number) for maternal Cas9 supply (heterozygous donor) and exogenous Cas9 supply as mRNA or protein (wild-type donor). B) Percentage of total embryos harbouring the desired point mutation knock-in allele for maternal Cas9 supply and exogenous Cas9 supply as mRNA or protein. Data inside the bars are the number of embryos sampled.

Fig 5. Indel and genomic deletion alleles identified in mutant pups generated from microinjection of sgRNA into fertilized zygotes derived from Cas9 expressing female mice.

Alleles identified in each mutant founder mice are aligned against the wild-type sequence. In homozygous mutant mice, only one allele is shown. In heterozygous alleles with an intact wild-type allele present, only the mutant allele is shown. In compound heterozygous mutant mice with two mutant alleles, both alleles are shown. Where more than two alleles, in addition to the wild-type sequence are present (mosaic founders), the mutant alleles identified are shown. Protospacers in the wild-type sequence are shown in red, with the PAM motif highlighted in blue. Where the mutated offspring had not inherited the Cas9 transgene, the entry in the table is marked with a grey background.

Fig 6. Alleles identified in mutant pups generated from microinjection of guide-RNA and ssODN into fertilized zygotes derived from Cas9 expressing female mice.

Alleles identified in each mutant founder mice are aligned against the wild-type sequence. In homozygous mutant mice, only one allele is shown. In heterozygous alleles with an intact wild-type allele present, only the mutant allele is shown. In compound heterozygous mutant mice with two mutant alleles, both alleles are shown. Where more than two alleles, in addition to the wild-type sequence are present (mosaic founders), the mutant alleles identified are shown. Protospacers in the wild-type sequence are shown in red, with the PAM motif highlighted in blue. For HDR ssODN templates the variant nucleotides are shown in green. Where the mutated offspring had not inherited the Cas9 transgene, the entry in the table is marked with a grey background.

Fig 7. Mutagenesis with cryopreserved embryos and using synthetic crRNA:tracrRNA.

(A) Target site amplification in blastocysts after microinjection of thawed zygotes with sgRNA. Zygotes had previously been prepared from heterozygous Cas9 expressing females and cryopreserved by vitrification. Doublets are indicative of indel mutagenesis (black arrows). (B) Percentage mutation rates (mutant embryos per total number) in either heterozygous (Cas9/+) or wild-type (WT) embryos obtained from heterozygous Cas9 expressing females, following microinjection with synthetic crRNA:tracrRNA alone. Data inside the bars are the number of embryos sampled.