Abstract
The International Mouse Phenotyping Consortium (IMPC) systematically produces and phenotypes mouse lines with presumptive null mutations to provide insight into gene function. The IMPC now uses the programmable RNA-guided nuclease Cas9 for its increased capacity and flexibility to efficiently generate null alleles in the C57BL/6N strain. In addition to being a valuable novel and accessible research resource, the production of 3,313 knockout mouse lines using comparable protocols provides a rich dataset to analyze experimental and biological variables affecting in vivo gene engineering with Cas9. Mouse line production has two critical steps – generation of founders with the desired allele and germline transmission (GLT) of that allele from founders to offspring. A systematic evaluation of the variables impacting success rates identified gene essentiality as the primary factor influencing successful production of null alleles. Collectively, our findings provide best practice recommendations for using Cas9 to generate alleles in mouse essential genes, many of which are orthologs of genes linked to human disease.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
↵¶ A list of authors and their affiliations appear at the end of this paper
The core analyses presented in this manuscript remain the same, but we have provided more explicit recommendations about optimizing genome editing experiments in mice. In addition, the information about the larger resource has been removed and will be presented in a separate manuscript in the future.
https://github.com/The-Centre-for-Phenogenomics/IMPC-Cas9-Production