Novel homology-directed gene targeting to enhance biomedical modeling

人类的健康和福祉受到生物医学进步的安全保护，生物医学进步往往需要对整个动物进行研究。这反映了这样一个事实，即我们对细胞--所有动物的构件--是如何发育、生长和发挥正常功能的，我们仍然只有有限的看法。许多常见的例子说明了这种全动物知识的本质：神经元不会患上帕金森氏症，胰腺细胞不会变得肥胖--人类确实会。归根结底，只有通过研究整个动物才能了解疾病和医学进步。考虑到这一点，人们往往希望只改变动物的一个特征或基因，看看对健康和疾病会有什么影响。通过这种方式，结果可以与被改变的特定特征或基因相关。问题是，进行这些指定的更改是耗时、不稳定、昂贵和低效的。二十多年来，研究人员一直在改变小鼠的特定基因--所谓的‘基因打靶’，但在这段时间里，同样的方法一直占据主导地位，在一些具有重要生物医学意义的大型物种中，这是令人望而却步的困难。基于我们在随机转基因方面的丰富经验，我们提出，一种新的精子注射方法可以实现高比例的定向DNA整合。这种方法使用了最近发现的引导RNA、CRISPR和称为Cas9的分子剪刀。然而，我们的Cas9方案与其他方案不同，后者在受精很长时间后将Cas9系统引入胚胎。相反，我们在受精发生的同时引入了Cas9系统。以这种方式生产基因组工程小鼠需要数周时间，不同于使用胚胎干细胞的传统多步方法，这需要长达一年的时间。如果建议的方法如我们的初步数据所显示的那样成功，它将比传统标准使用的动物少65%，效率将是其他最好的基于Cas9的方法的3倍。新方法将如何工作？简而言之，它利用了受精的一个独特特征：精子一进入卵子，其遗传物质(DNA基因组)就被拆开，可以与其他DNA重组。通过在受精的同时引入Cas9系统，Cas9分子剪刀可以在所需位置快速切割暴露的精子基因组。如果我们同时引入一段量身定做的DNA，以匹配切割的每一边，它将整合在一起，从而产生的后代中的每个细胞都包含量身定做的片段。如果成功，这种方法将允许我们插入大块的DNA片段，定位到典型哺乳动物基因组中约30亿个碱基的一个位置。我们预计的结果是，新生儿后代的每个细胞都将包括定制的改变。因为这个想法是新的，它必须在一个易于驯服的哺乳动物模型系统中发展出来，为此我们使用了小鼠。老鼠是人类疾病建模工具包的关键组成部分，但我们预计所取得的进展也将适用于其他生物医学上的重要物种，如猪。因此，建议的方法在基因靶向极其困难或不可能的大型生物医学模式物种中的应用将是极其重要的。我们在其他类型的基因组操作方面的经验表明，如果一种方法在小鼠身上有效，它也适用于更大的物种，从而减少所需的数量，并节省相当大的经济成本。因此，这项提议是及时的，并有望简化生物医学研究，使模型的生产能够评估疾病、基于干细胞的治疗和异种移植，从而加快下一代诊断和治疗药物的交付。

Human health and well-being is safe-guarded by biomedical advances which often require research on whole animals. This reflects the fact that we still have only a limited view of how cells - the building blocks of all animals - develop, grow and perform their normal functions. The essential nature of this whole-animal knowledge is illustrated with some of the many commonly-encountered examples: neurons do not contract Parkinson's Disease and pancreatic cells do not become obese - people do. Ultimately, it is by studying whole animals that diseases will be understood and medical advances achieved.With this in mind, it is often desirable to change only one trait or gene in an animal to see what the consequences are in health and disease. In this way, the outcome can be related to the specific trait or gene that has been altered. The problem is that making these specified changes is time-consuming, unstable, expensive and inefficient. Researchers have been altering specified genes - so-called 'gene targeting' - in mice for well over two decades, but the same method has predominated in all that time and in some biomedically important large species it is prohibitively difficult.Based on our extensive experience with random transgenesis, we propose that high rates of targeted DNA integration are achievable by a novel sperm injection method. This method employs the recently discovered guide RNA, CRISPR, and molecular scissors called Cas9. However, our Cas9 protocol is different from others, which introduce the Cas9 system into embryos long after fertilisation. Instead, we introduce the Cas9 system at the same time that fertilisation occurs. Producing genome-engineered mice in this way should take weeks, unlike the traditional multi-step method using embryonic stem (ES) cells, which takes up to a year. If the proposed method is as successful as our preliminary data suggest, it will use 65% fewer animals than the conventional standard and will be 3x more efficient than the best other Cas9-based methods.How will the new method work? In brief, it harnesses a unique feature of fertilisation: as soon as the sperm enters the egg, its genetic material (the DNA genome) is unpackaged and is available to recombine with other DNA. By introducing the Cas9 system at the same time as fertilisation, the Cas9 molecular scissors rapidly cut the exposed sperm genome at the desired position. If at the same time we introduce a segment of DNA tailored to match each side of the cut, it will integrate so that every cell in the resulting offspring contains the tailor-made segment. If successful, the method will allow us to insert large pieces of DNA pin-pointed to one position in the 3 billion or so bases in a typical mammalian genome. The result we expect is that every cell of the newborn offspring will include the bespoke alteration.Because this idea is new, it must be developed in a tractable mammalian model system, for which we use the mouse. The mouse is a critical component of the human disease modeling toolkit, but we expect that the advances made will also be applicable to other biomedically important species such as pigs. The utility of the proposed method in large biomedical model species, where gene-targeting is extremely difficult or impossible, will therefore be enormously important. Our experience with other types of genome manipulation has shown that if a method works in the mouse, it also works in larger species, serving to reduce the numbers required with considerable economic savings. This proposal is accordingly timely and promises to streamline biomedical research, enabling the production of models to evaluate disease, stem cell-based therapies and xenotransplantation and thereby accelerate the delivery of next-generation diagnostic and therapeutic medicine.