Generation of mutant mice by pronuclear injection of circular plasmid expressing Cas9 and single guided RNA

Abstract

CRISPR/Cas mediated genome editing has been successfully demonstrated in mammalian cells and further applications for generating mutant mice were reported by injecting humanized Cas9 (hCas) mRNA and single guide RNA into fertilized eggs. Here we inject the circular plasmids expressing hCas9 and sgRNA into mouse zygotes and obtained mutant mice within a month. When we targeted the Cetn1 locus, 58.8% (10/17) of the pups carried the mutations and six of them were homozygously mutated. Co-injection of the plasmids targeting different loci resulted in the successful removal of the flanked region in two out of three mutant pups. The efficient mutagenesis was also observed at the Prm1 locus. Among the 46 offspring carrying CRISPR/Cas plasmid mediated mutations, only two of them carried the hCas9 transgene. The pronuclear injection of circular plasmid expressing hCas9/sgRNA complex is a rapid, simple, and reproducible method for targeted mutagenesis.

Figure 1. Scheme for CRISPR/Cas mediated gene manipulation.

(a) pCAG-EGxxFP plasmid contains 5′ and 3′ EGFP fragments that shares 482 bp under ubiquitous CAG promoter. The ~500 bp genomic fragment containing the sgRNA target sequence was placed between EGFP fragments of pCAG-EGxxFP plasmid. The resulting target plasmid was cotransfected with pX330 plasmids expressing sgRNA and hCas9 into HEK293T cells. When the target sequence was digested by sgRNA guided CAS9 endonuclease, the homology dependent repair (HR; homologous recombination or SSA: single strand annealing) took place and reconstituted the EGFP expression cassette. MCS; multi cloning sites. (b) The plasmids used in the study. pCAG-EGxxFP contains multicloning sites (BamHI, NheI, PstI, SalI, EcoRI, and EcoRV). pX330 and pT7-sgRNA plasmids contains BbsI sites that enables directional cloning of sgRNA oligos. (c) The efficiency of DSB mediated homology dependent repair was validated by observing EGFP fluorescence 48 hrs after the transfection (top; pX330 without sgRNA, bottom; pX330 with Cetn1/sgRNA1). (d) To generate gene disrupted mice, fertilized eggs were injected with RNAs coding hCas9 and sgRNA into cytoplasm or pX330 plasmid into pronuclei.

Figure 2. CRISPR/Cas mediated Cetn1 and Prm1 mutations in mice.

(a) Cetn1 mutations observed in founder mice. The small indels were identified at sgRNA targeted locus (bold; sgRNA recognition site, red; PAM sequence, slash; predicted cleavage site, and arrow; sgRNA direction). Some mutations were frequently observed in independent founder mice. The numbers of mutants obtained were indicated in parenthesis (left; with plasmid injection, right; with RNA injection). (b) Representative Cetn1 genomic sequences from founder mice (top; wild type, middle; heterozygous 8 bp deletion, homozygous 8 bp deletion). (c and d) Prm1 mutations observed in founder mice.

Figure 3. Male infertility found in Cetn1 and Prm1 deficient mice.

(a) Average litter size obtained from Cetn1 mutant males. Each male was mated with two B6D2F1 females for two months and numbers of pups were counted at birth. (b) Sperm collected from epidydimis of 12 weeks old males were photographed under a phase contrast microscopy. Impaired ciliogenesis was observed in Cetn1^em4/em4 mice. Scale bar; 20 um. (c) Sperm motility observed with Prm1 mutant males. The eggs were collected from superovulated B6D2F1 females mated with 12 weeks old males. The sperm motility was anlayzed by Ceros system at 10 min and 120 min of incubation. (d) Sperm collected from epidydimis of 12 weeks old males were photographed under a differential interference contrast microscopy. Heads narrowed and reduced in curvature at the tip were observed in most Prm1^+/em3 sperm. Scale bar; 20 um.