Reducing the animal cost of CRISPR/Cas9 mutagenesis

Lead Research Organisation: University of Oxford
Department Name: Wellcome Trust Centre for Human Genetics

Abstract

Genetically altered mouse models allow scientists to investigate the function of genes and their role in particular biological processes. Although, a great deal of information can be gained by studying the role of genes in cell culture systems, when a gene plays a role in complex biology, for example influencing behaviour or immune response, animal models are necessary to fully explore gene function in a whole animal.
By generating mouse models where a particular gene has been removed, the role of the missing gene can be investigated by examining the mutant mouse closely and establishing how it differs from normal mice with the gene present. Similarly, specific mutations, which, in humans, are known or suspected to be responsible for genetic disease, can be introduced into the equivalent position in the mouse. These genetically altered mouse models are being used to investigate the underlying cause of the disease process and to trial novel therapeutic and diagnostic approaches.
In the last few years, the way scientists generate mouse models has changed dramatically. Enzymes that cut specific DNA sequences can now be generated in the laboratory and can be introduced into the mouse embryo. The resulting break in the DNA can be exploited to either mutate or introduce specific changes in the DNA. This has made the process of generating genetically altered mouse models considerably faster, easier and cheaper. Many new mouse models are being generated and we anticipate seeing this technology increase the number of animal experiments performed around the world.
Despite its wide application, the true animal cost of the new technology remains unexplored. Already clear problems have been identified. Firstly, the mice that are generated from an injection of these DNA-cutting enzymes are frequently complex, undefined mosaics of many different types of mutation - that is to say, that different cells within the mouse can carry different mutations. This is because the enzymes injected into the 1-cell embryo remain active for a long time and can persist after the embryo divides into the 2-cell, 4-cell and later stage embryo, cutting and re-cutting the genome leading to different mutations in different cells. A large amount of breeding may be required to generate offspring from this mosaic mouse that have the required mutation. Furthermore, this mosaic outcome prevents the analysis of the first generation and necessitates the breeding of animals.
We plan to investigate ways of restricting the activity of the nucleases so that they are no longer active after the first division of the 1-cell embryo. If successful, this would eliminate the frequent mosaicism seen and avoid much of the downstream breeding of mice. This improvement could lead to a situation where, in certain instances, for example for preliminary screening, the first generation can be directly assessed, thus avoiding the need to maintain colonies of mice altogether - leading to a reduction in mouse usage for in vivo functional gene analysis.
An additional problem with the new technology is that the enzymes are very active and frequently mutate both copies of a target gene. Sometimes this is the desired outcome, but on other occasions, in particular when the aim of the experiment is to introduce a specific mutation into a target gene, a deleterious mutation on the other copy of a gene can result, which can lead to more severe consequences for the animal. Our research aims at investigating ways of avoiding this phenomenon, allowing mutation of only one of the two copies of a gene. If successful, this could represent an important refinement, as any harmful effects of gene mutation would be alleviated.

Technical Summary

Genetically altered (GA) mouse models provide a powerful means of attributing function to DNA sequences. As investigations of how DNA variation and mutation contributes to disease advance, demand for GA mouse models increases. Recently, this demand has been aided by the new CRISPR/Cas9 technology, an RNA guided nuclease system, which permits the production of models at unprecedented efficiencies.
Already the biomedical community is applying this new technology widely for mouse model production. Meanwhile, the 3Rs impact of the technology remains unexplored. This proposal seeks to address aspects of the technology that, our experience shows, can lead to substantial increases in mouse usage and wasted production of undesirable genotypes.
Following CRISPR/Cas9 microinjection into mouse zygotes, the founders generated are invariably mosaic, due to the persistence of the nuclease after the first cleavage event. Founders must be bred extensively to ensure transmission of the desired allele, increasing mouse usage. The mosaicism also precludes the phenotyping of the founder generation, which, given the high efficiencies of CRISPR/Cas9 might be feasible for certain phenotypic screens.
We plan to explore whether the use of Cas9 fusions with destabilizing or cell-cycle regulatory domains can be used to confine nuclease activity to the 1-cell stage and thus eliminate mosaicism. Furthermore, the mode of Cas9 supply, protein or maternally contributed via transgenic Cas9 overexpression will be investigated for its impact on mosaicism.
As a potential refinement, methods to address and limit deleterious indel mutagenesis during knock-in mice production, a class of model particularly sought after for interrogating disease-related mutation, will be investigated. Whether the mode of nuclease delivery, the use of cell cycle-regulated Cas9 and co-injection of wild-type repair templates can impact the rate of indel mutation at the non-targeted allele will be assessed.

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