new mouse models Embryology
Since 06/2017
Scarless introduction of disease-associated variant in mouse (12/2017)
We successfully used CRISPR + ssOligo donor approach to mutate only a single nucleotide without leaving any other unwanted mutations behind (scarless editing). The point mutation was aimed within the 5'UTR of a gene to convert G to A (identified splice site) thereby mimicking a human disease mutation in mouse. Because the introduction of silent mutations was not desirable the gRNA binding site had to span the mutation site in order to minimize the chance of re-cutting of Cas9 after successful integration of the ssOligo donor and all pups needed to be screened by sanger sequencing to check for the presence of the mutation.
A) Design overview. gRNA was chemically synthesised as crRNA and annealed with tracrRNA followed by pre-complexing with Cas9 protein (RNP) and co-injected with ssOligo donor into the cytoplasm of mouse zygotes. B) FO pups were screened by PCR followed by sanger sequencing to identify positive pups (#7, 13). C) Positive clones were TOPO cloned and 12 colonies sequence verified to check DNA sequence on both alleles.
Generation of a KI mouse harboring a point mutation (11/2017)
We successfully used CRISPR + ssOligo donor approach to introduce a point mutation. Two gRNAs were chosen that would enable Cas9 to cut the genomic DNA in close proximity to the insertion site. The point mutation was aimed within an exon to convert Arginine (R) to Alanine (A). This can be achieved by changing the corresponding triplet CGG to GCT (red). In addition, two silent mutations (green) were introduced to create a novel restriction site (SacI). This served two purposes: (i) to enable screening of F0 pups by restriction analysis and (ii) to destroy the gRNA binding site to avoid re-cutting of Cas9 after successful integration of the oligo donor.
A) Design overview. Each gRNA was chemically synthesised as crRNA and annealed with tracrRNA followed by pre-complexing with Cas9 protein (RNP) and co-injected with ssOligo donor into the cytoplasm of mouse zygotes. B) FO pups were screened by PCR followed by SacI digestion to identify positive pups (red asterisk). C) Positive clones were TOPO cloned and 12 colonies sequence verified to check DNA sequence on both alleles.
Generation of a KO mouse by exon deletion (08/2017)
We successfully used CRISPR to generate a KO mouse. Here, 4 gRNAs (two on either site) were chosen that flank the "critical exon" (c.e.). Successful removal of the critical exon leads to a frameshift in the downstream coding sequence and therefore to a premature stop codon (red). Homozygous F0 pups were backcrossed to WT mice with all F1 mice being heterozygous thereby confirming germline transmission of the KO allele.
A) Design overview. Each gRNA was chemically synthesised as crRNA and annealed with tracrRNA followed by pre-complexing with Cas9 protein (RNP) and injected into the cytoplasm of mouse zygotes. FO pups were screened by PCR to identify positive founder.
B) Positive clones were TOPO cloned and 12 colonies sequence verified to check DNA sequence on both alleles.
WT: Wild-type
HT: Heterozygous
HO: Homozygous
ES cell injection into Blastocyts (03/2017)
We successfully used ES cells to generate a new mouse line. Germline Transmission was confirmed by coat colour.