Novel homology-directed gene targeting to enhance biomedical modeling

新型同源基因靶向增强生物医学模型

• 批准号: MR/N020294/1
• 负责人: Anthony Perry
• 金额: $ 44.65万
• 依托单位: University of Bath
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2016
• 资助国家: 英国
• 起止时间: 2016 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FN020294%2F1
• 关键词: Novel; homology; directed; gene; targeting

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.

人类的健康和福祉受到生物医学进步的保障，这些进步往往需要对整个动物进行研究。这反映了一个事实，即我们对细胞-所有动物的基石-如何发育，生长和执行其正常功能仍然只有有限的看法。这种全动物知识的本质是用许多常见的例子来说明的：神经元不会患帕金森病，胰腺细胞不会变胖--人会。最终，只有通过研究整个动物，才能了解疾病，实现医学进步。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，看看对健康和疾病的影响。通过这种方式，结果可以与已经改变的特定性状或基因相关。问题是，进行这些指定的更改是耗时的，不稳定的，昂贵的和低效的。二十多年来，研究人员一直在改变小鼠的特定基因--所谓的“基因靶向”，但在这段时间里，同样的方法一直占主导地位，而在一些生物医学上重要的大型物种中，这是非常困难的。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速率的靶向DNA整合。这种方法使用了最近发现的指导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，其他方案在受精后很长时间内将Cas9系统引入胚胎。相反，我们在受精发生的同时引入Cas9系统。以这种方式生产基因组工程小鼠需要数周时间，而传统的使用胚胎干细胞（ES）的多步骤方法需要长达一年的时间。如果该方法像我们的初步数据所显示的那样成功，它将比传统标准少使用65%的动物，并且比其他最好的基于Cas9的方法效率高3倍。简而言之，它利用了受精的一个独特特征：一旦精子进入卵子，其遗传物质（DNA基因组）就会被解开，并可与其他DNA重组。通过在受精的同时引入Cas9系统，Cas9分子剪刀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与切割的每一侧相匹配的DNA片段，它将整合，这样产生的后代中的每个细胞都包含定制的片段。如果成功的话，这种方法将使我们能够在典型的哺乳动物基因组中30亿左右的碱基中插入大片段的DNA。我们期望的结果是新生后代的每个细胞都包含定制的改变，因为这个想法是新的，它必须在一个易于处理的哺乳动物模型系统中开发，我们使用小鼠。小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重要物种，如猪。因此，在基因靶向极其困难或不可能的大型生物医学模型物种中，所提出的方法的实用性将非常重要。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中有效，那么它也适用于更大的物种，从而减少了所需的数量，节省了可观的经济成本。因此，这项提案是及时的，并有望简化生物医学研究，使模型的生产，以评估疾病，干细胞治疗和异种移植，从而加快下一代诊断和治疗药物的交付。

项目成果

Development of mammalian cell logic gates controlled by unnatural amino acids.

• DOI: 10.1016/j.crmeth.2021.100073
• 发表时间: 2021-10-25
• 期刊: Cell reports methods

Human embryonic genome activation initiates at the one-cell stage.

• DOI: 10.1016/j.stem.2021.11.012
• 发表时间: 2022-02-03
• 期刊: Cell stem cell
• 影响因子: 23.9
• 作者: Asami M;Lam BYH;Ma MK;Rainbow K;Braun S;VerMilyea MD;Yeo GSH;Perry ACF
• 通讯作者: Perry ACF

Genomic imprinting in mouse blastocysts is predominantly associated with H3K27me3.

• DOI: 10.1038/s41467-021-23510-4
• 发表时间: 2021-06-21
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Santini L;Halbritter F;Titz-Teixeira F;Suzuki T;Asami M;Ma X;Ramesmayer J;Lackner A;Warr N;Pauler F;Hippenmeyer S;Laue E;Farlik M;Bock C;Beyer A;Perry ACF;Leeb M
• 通讯作者: Leeb M

Intracytoplasmic Sperm Injection

胞浆内单精子注射

• DOI: 10.1007/978-3-319-70497-5_13
• 发表时间: 2018
• 作者: Griffin D

Switchable genome editing via genetic code expansion.

可切换基因组编辑通过遗传代码扩展。

• DOI: 10.1038/s41598-018-28178-3
• 发表时间: 2018-07-03
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Suzuki T;Asami M;Patel SG;Luk LYP;Tsai YH;Perry ACF
• 通讯作者: Perry ACF