Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for correcting variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations

Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines

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

Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations

Impact of essential genes on the success of genome editing experiments generating 3313 new genetically engineered mouse lines.

Acknowledgements: We thank all technical personnel at the IMPC production centres for their contributions. H.E., E.A., M.G., L.L., C.M., and L.M.J.N. were supported by Ontario Genomics and Genome Canada OGI-051, OGI-090, OGI-137 and the Canada Foundation for Innovation. M-C.B. and Y.H. were supported by the Université de Strasbourg, the CNRS, the INSERM and the ‘Investissements d’avenir’ programs (ANR-10-IDEX-0002-02, ANR-10-LABX-0030-INRT and ANR-10-INBS-07 PHENOMIN). A.C., G.C., M.M., L.T., and S.W. were supported by the Medical Research Council MC_UP_1502/3 International Mouse Phenotyping Consortium—building a functional catalogue of a mammalian genome. B.D., G.D., E.R., H.W.-J., D.A., A.B., R.R.-S., and W.S. were supported by the Wellcome Trust. P.M. and H.P. were supported by European Molecular Biology Laboratory core funding. P.K. and R.S. used services of the Czech Centre for Phenogenomics supported by the Czech Academy of Sciences RVO 68378050 and project LM2018126 Czech Centre for Phenogenomics provided by Ministry of Education, Youth and Sports of the Czech Republic, LM2015040 Czech Centre for Phenogenomics by MEYS, CZ.1.05/2.1.00/19.0395 Higher quality and capacity for transgenic models by MEYS and ERDF, CZ.1.05/1.1.00/02.0109 Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University in Vestec (BIOCEV) by MEYS and ERDF, CZ.02.1.01/0.0/0.0/16_013/0001789 Upgrade of the Czech Centre for Phenogenomics by MEYS and ESIF. Research reported in this publication was supported by the NIH Common Fund, the Office of The Director and the National Human Genomic Research Institute of the National Institutes of Health (U42OD011174 and UMIHG006348 supported A.C., D.L., G.C., F.D., I.L., M.M., J.S., L.T., S.W., M.D., and J.D.H.; U42OD011175 and UM1OD023221 supported M.G., L.L., C.M., L.M.J.N., B.W., J.A.W., M.R., and K.L.; U42OD011185 and UM1OD023222 supported L.G., K.P., R.B., J.K.W., and S.A.M.; UM1HG006370 supported A.-M.M. and H.P.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Funder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100010269The 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

Extensive identification of genes involved in congenital and structural heart disorders and cardiomyopathy

Clinical presentation of congenital heart disease is heterogeneous, making identification of the disease-causing genes and their genetic pathways and mechanisms of action challenging. By using in vivo electrocardiography, transthoracic echocardiography and microcomputed tomography imaging to screen 3,894 single-gene-null mouse lines for structural and functional cardiac abnormalities, here we identify 705 lines with cardiac arrhythmia, myocardial hypertrophy and/or ventricular dilation. Among these 705 genes, 486 have not been previously associated with cardiac dysfunction in humans, and some of them represent variants of unknown relevance (VUR). Mice with mutations in Casz1, Dnajc18, Pde4dip, Rnf38 or Tmem161b genes show developmental cardiac structural abnormalities, with their human orthologs being categorized as VUR. Using UK Biobank data, we validate the importance of the DNAJC18 gene for cardiac homeostasis by showing that its loss of function is associated with altered left ventricular systolic function. Our results identify hundreds of previously unappreciated genes with potential function in congenital heart disease and suggest causal function of five VUR in congenital heart disease