PgmNr M314: Cas9 RNA-guided nuclease gene editing – rapid disease modeling in mice.

Authors:
L. M. J. Nutter ^1,2 ; J. Ellegood ³ ; M. Gertsenstein ² ; A. Flenniken ^2,4 ; R. M. Henkelman ¹ ; C. McKerlie ^1,2 ; J. P. Lerch ¹ ; J. Rossant ¹

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Institutes
1) The Hospital for Sick Children, Toronto, ON, CA; 2) The Centre for Phenogenomics (TCP), Toronto, ON, CA; 3) Mouse Imaging Centre, Toronto, ON, CA; 4) 4Lunenfeld-Tanenbaum Research Institute, Toronto, ON, CA.

Abstract:

Cas9 RNA-guided nuclease (CRISPR/Cas9) gene editing is revolutionizing genetic modification in many species. The Model Production Core at The Centre for Phenogenomics (TCP) is a national facility serving academia and industry in Canada and internationally. TCP has implemented CRISPR/Cas9 editing in mice to produce knockout mutations for the International Mouse Phenotyping Consortium as well as knockout and point mutant alleles for investigators seeking to model specific human diseases.

We produced models by co-injecting mouse zygotes with target-specific guide RNA(s), Cas9 endonuclease, and when required, a homologous repair template. We have designed and produced multiple lines with disease-associated point mutations for mechanistic and pre-clinical studies as well as knockout mouse lines for both medium- and high-throughput phenotype screens. Our success rate for requested alleles is >80% at >50 different loci in the C57BL/ 6NCrl background. No off-target mutagenesis was detected for 12 different gRNAs in 93 N1 mice for 852 potential off-target loci. However, real-time quantitative PCR for repair template copy number has identified multi-copy insertions in some mice. Thus, while off-target mutagenesis appears to be a very low risk with locus-specific guide RNAs, quality control should include copy number screens for repair templates when they are used.

We set out to compare phenotypes of ES cell-derived and Cas9-derived mutant lines; assess novel mutants for neuroanatomical phenotypes; and produce mouse models of human disease. We produced two Tox3 knockout mouse lines; one from targeted ES cells and the other using Cas9, C6N-Tox3^/MbpCrl and C6N-Tox3^em1Tcp, respectively. For phenotyping, we intercrossed heterozygotes within each line. Homozygotes for each of these alleles both died before 4 weeks of age and had reduced brain volume. We also used Cas9 to produce several knockout mouse lines in genes associated with Autism Spectrum Disorder as part of a medium-throughput screen to identify neuroanatomical phenotypes. We show that many of these mutants had both in vivo behavioural phenotypes as well as brain dysmorphology compared to wild type. Finally, we have produced and delivered several disease-associated point mutants that are currently under study. One of these presents with seizures, a clinical sign observed in the patient population. This mutant is now being used to assess hyperexcitability in cortical neurons as well as in a drug screen.

In conclusion, Cas9 mutagenesis is an efficient way to produce mutant mouse lines at high throughput with specific gene mutations for modeling human disease.