The creation of targeted mutations by the use of CRISPR/Cas9 gene editing has revolutionised the process and possibilities of altering the function of genes in a wide variety of animals and plants. We describe the challenges of transitioning this technology into a high-throughput mouse production environment with the generation and characterisation of over one hundred new mutant mouse strains.
The majority of our CRISPR-mediated knockout strains are the result of whole exon deletions in which two guide RNAs are designed per flanking region to maximise the chance of a successful double-strand break. The gRNAs and Cas9 mRNA are introduced into 1-cell mouse zygotes by cytoplasmic injection and the G0 mice screened by a rapid PCR/qPCR method to determine the level of mosaicism and the percentage of putative deletion events. The mutation is then sequence-verified in the next generation to ensure that complete deletion of the exon has occurred.
We have found that the use of the CRISPR/Cas9 system has significant advantages in both the speed and level of generation of new strains, and has allowed us to significantly reduce the numbers of animals needed to achieve germline transmission.