Novel homology-directed gene targeting to enhance biomedical modeling

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.

Unfertilized mouse eggs will be injected with a sperm (ICSI), CRISPR (gRNA) and Cas9 genome engineering components, plus an exogenous construct to promote targeted DNA integration by homology-directed repair (HDR). This approach harnesses sperm chromatin decondensation during fertilisation; decondensation depletes the paternal genome of almost all nucleoprotein, making it an extremely good substrate for recombination.

Previous methods to engineer whole animals inject the Cas9 machinery into embryos that have long-since completed fertilisation, skipping this recombinogenic decondensation step. We have shown that sperm injection harnesses decondensation to enhance recombination in random transgenesis and homology-independent Cas9-mediated editing. We now seek to optimise DNA integration by HDR in a sperm injection protocol that we have already demonstrated in preliminary experiments.

We will demonstrate targeted DNA integration following sperm injection with CRISPR, Cas9 cRNA and proven targeting constructs. These target Oct4, Rex1 or Cdx2 genes, resulting in characteristic fluorescence patterns in preimplantation embryos after culture in vitro for 3.5 days. Gene targeting in embryos with promising expression patterns will be confirmed by RT-PCR and genomic PCR, which we routinely apply to preimplantation embryos. This in vitro system allows us to optimise the protocol without having to produce offspring, cutting costs, time and animal numbers.

The method promises to streamline mouse and large animal genome-engineered biomedical modeling. We will demonstrate targeting in mouse offspring by transferring embryos after micromanipulation to recipient surrogate mothers. Offspring will be characterized phenotypically, to confirm that the method does not introduce unexpected phenotypes, and genetically by PCR and Southern blotting to reveal the percentage of offspring harbouring alleles with the anticipated targeted configuration.

1. The 3Rs in mouse models of human disease
The protocol we propose is conservatively expected to reduce by 65% the number of animals required to produce a gene-targeted line by conventional methods, representing an annual saving to the UK of 282,000 mice at present rates. This reduction is possible because the proposed method: (i) circumvents lengthy breeding programmes, (ii) will be more efficient than existing methods, (iii) will enable 'dead-end' phenotypes to be identified rapidly in the life of a project, eliminating unnecessary breeding.

2. Better and easier large animal biomedical research tools
The targeting method promises to have an enormous impact on large mammal genome engineering which is at present challenging, expensive and unreliable. Pig genome engineering will in all likelihood prove indispensible to next generation human medicine in three ways: (i) for evaluating stem cell therapeutics prior to Phase I clinical trials where mouse models are inadequate, for example because their lifespan is too short or there are insurmountable physiological barriers, (ii) for producing superior disease models, for example in neurodegenerative or other chronic disease, and (iii) for pig-to-human xenotransplantaton. However, there are no verified pig ES cell lines and pig targeted DNA integration has been achieved by nuclear transfer, an incredibly non-trivial method. Porcine genome engineering during sperm injection described here would reduce by years the time taken to generate each targeted line. For example, the idea that pigs might be universal organ donors in pig-to-human xenotransplantation was derailed 15 years ago in large part because of fears that porcine endogenous retroviruses (ERVs) could skip the species barrier (zoonosis) following transplantation. Pig genome engineering promises to eliminate ERV zoonotic transmission by excising all proviral integrants; pigs contain between 0 and 50 depending on the breed.

3. Clinical impacts: infertility, cancer and regenerative medicine
Infertility. We will use the method to produce models of impaired fertility. This will contribute to assisted reproductive technology (ART) treatment and diagnosis, benefiting healthcare workers and many of the 3.5 million people in the UK who have difficulty conceiving, impacting commercial entities conducting ART, such as the CARE Fertility, and UK charities such as WellBeing of Women.
Cancer. Improved complex genome targeting will benefit clinical oncologists and patients by creating pipelines to recapitulate disease and evaluate therapy. Promising results will be translated to pigs.
Regenerative medicine. Facilitating large and small animal models of safe cellular potency will increase the efficacy of cellular medicine and help move it from bench to bedside. This coheres with Aim One of the MRC strategic plan by enabling safe regenerative therapies and will translate into a competitive advantage for commercial entities such as the UK Stem Cell Foundation.

4. UK knowledge transfer and development
Following relocation of the lab from Japan, the proposal is based on our demonstration of genome targeting using technologies not widely established outside Asia. These technologies will not only impact genome engineering but have broad trans-disciplinary impacts. For example, we are developing novel nanodevices in collaboration with physicists at the Microelectronics Institute of Barcelona IBM-CNM. Intracellular machines will complement genome engineering for a better understanding of disease pathways. Allied technologies from the Perry lab will facilitate mouse strain archiving. The explosive advance in Cas9 research is already transforming the multi-million dollar international gene targeting market, so this work will be of interest to genome engineering biotechnology companies. The method will have commercial value both as a technical improvement and by offering legal alternatives to current IP.