Reducing the animal cost of CRISPR/Cas9 mutagenesis

转基因小鼠模型使科学家能够研究基因的功能及其在特定生物过程中的作用。虽然，通过研究基因在细胞培养系统中的作用可以获得大量信息，但当基因在复杂的生物学中发挥作用时，例如影响行为或免疫反应，动物模型是必要的，以充分探索整个动物的基因功能。通过建立特定基因被移除的小鼠模型，可以通过仔细检查突变小鼠并确定它与存在该基因的正常小鼠有何不同来研究缺失基因的作用。类似地，在人类中已知或怀疑导致遗传病的特定突变可以被引入到小鼠的相同位置。这些转基因小鼠模型正被用于调查疾病过程的根本原因，并试验新的治疗和诊断方法。在过去的几年里，科学家制作老鼠模型的方式发生了戏剧性的变化。切割特定DNA序列的酶现在可以在实验室产生，并可以被引入小鼠胚胎。由此产生的DNA断裂可以被利用来突变或在DNA中引入特定的变化。这使得产生转基因小鼠模型的过程变得更快、更容易、更便宜。许多新的小鼠模型正在产生，我们预计这项技术将增加世界各地进行的动物实验的数量。尽管它得到了广泛的应用，但这项新技术的真实动物成本仍未得到探索。已经发现了明显的问题。首先，注射这些DNA切割酶产生的小鼠往往是许多不同类型突变的复杂、未定义的马赛克-也就是说，小鼠体内的不同细胞可以携带不同的突变。这是因为注入到1-细胞胚胎中的酶在很长一段时间内保持活跃，并在胚胎分裂为2-细胞、4-细胞和后期胚胎后持续存在，切割和重新切割基因组导致不同细胞中的不同突变。可能需要进行大量的繁殖才能从这种具有所需突变的嵌合体小鼠中产生后代。此外，这种马赛克结果阻碍了对第一代的分析，并需要繁殖动物。我们计划研究限制核酸酶活性的方法，使它们在1-细胞胚胎的第一次分裂后不再活跃。如果成功，这将消除频繁出现的嵌合体，并避免大量老鼠的下游繁殖。这种改进可能会导致一种情况，在某些情况下，例如在初步筛选时，第一代可以被直接评估，从而避免了完全保持小鼠群体的需要-导致用于体内功能基因分析的小鼠使用量减少。新技术的另一个问题是，酶非常活跃，经常突变目标基因的两个拷贝。有时这是想要的结果，但在其他情况下，特别是当实验的目的是将特定的突变引入目标基因时，可能会导致基因的另一个副本上的有害突变，这可能会给动物带来更严重的后果。我们的研究旨在研究避免这种现象的方法，只允许基因的两个副本中的一个发生突变。如果成功，这可能是一项重要的改进，因为基因突变的任何有害影响都将得到缓解。

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.