Novel homology-directed gene targeting to enhance biomedical modeling

点击翻译按钮获取中文摘要

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.