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.

CNRS INSERM CELPHEDIA PHENOMIN Institut Clinique de la Souris Université de Strasbourg Illkirch Graffenstaden France

Czech Centre for Phenogenomics Institute of Molecular Genetics of the Czech Academy of Sciences Vestec Czech Republic

Department of Integrative Physiology Baylor College of Medicine Houston TX 77030 USA

Department of Mathematics and Statistics University of Guelph Guelph ON N1G 2W1 Canada

Department of Molecular and Cellular Biology Baylor College of Medicine Houston TX 77030 USA

Department of Molecular and Human Genetic Baylor College of Medicine Houston TX 77030 USA

Department of Statistics University of Manitoba Winnipeg MB R3T 2N2 Canada

Department of Surgery School of Medicine University of California Davis Davis CA 95618 USA

European Molecular Biology Laboratory European Bioinformatics Institute Wellcome Genome Campus Hinxton UK

LGC Assure Fordham CB7 5WW UK

Mouse Biology Program University of California Davis Davis CA 95618 USA

MRC Harwell Institute Harwell OX11 0RD UK

Nuffield Department of Population Health University of Oxford Oxford OX3 7LF UK

Program in Craniofacial Biology and Department of Orofacial Sciences University of California San Francisco San Francisco CA USA

Reproductive and Developmental Biology Laboratory NIEHS Research Triangle Park Durham NC 27709 USA

The Centre for Phenogenomics Toronto ON M5T 3H7 Canada

The Francis Crick Institute 1 Midland Rd London NW1 1AT UK

The Hospital for Sick Children Toronto ON M5G 1X8 Canada

The Jackson Laboratory Bar Harbor ME 04609 USA

The Jackson Laboratory for Genomic Medicine Farmington CT 06032 USA

The Mary Lyon Centre MRC Harwell Institute Harwell Campus Didcot Oxon OX11 0RD UK

UT Health San Antonio San Antonio TX 78229 USA

Wellcome MRC Cambridge Stem Cell Institute University of Cambridge Trinity Lane Cambridge CB2 1TN UK

Wellcome Sanger Institute Wellcome Genome Campus Hinxton Cambridge CB10 1SA UK

William Harvey Research Institute Queen Mary University of London London EC1M 6BQ UK

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