Publication Date
9-30-2024
Journal
Scientific Reports
DOI
10.1038/s41598-024-72418-8
PMID
39349521
PMCID
PMC11443006
PubMedCentral® Posted Date
9-30-2024
PubMedCentral® Full Text Version
Post-print
Published Open-Access
yes
Keywords
Animals, Genes, Essential, Mice, Gene Editing, Mice, Knockout, CRISPR-Cas Systems, Alleles, Mice, Inbred C57BL, Male, Female, Genetic Engineering, Phenotype, Cas9, Genome editing, Mouse, Genetics, Genetic engineering
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 3313 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.
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Endocrine System Diseases Commons, Endocrinology, Diabetes, and Metabolism Commons, Genetic Processes Commons, Genetics Commons, Medical Genetics Commons
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