Developing an in vivo CRISPR-interference Screening Resource

A revolution in basic biological and medical research is currently taking place. The development of fast and inexpensive ways of reading DNA code, or sequencing, have led to a flood of information which can be used to identify the causes of all kinds of diseases and answer other important biological questions. The information generated by DNA sequencing often takes the form of very long lists of genes that are good candidates for either causing a disease, changing the outcome of a disease, or that may be controlling a biological process of interest. These gene lists, frequently 10-100 in length, only suggest "potential" candidates because high throughput approaches by themselves, do not "prove" a role for a gene in a particular process.

To prove involvement, we need to be able to increase or reduce the activity of the candidate gene and put its influence to the test. Although this can be done quite efficiently in cultured cells, many questions cannot be answered in this way because cultured cells often cannot accurately replicate what is happening in an animal. Very often, the context of a whole animal is essential.
In addition, some genes may control important functions in one part of the body, but control different functions in another. This means that we need an approach that only alters gene function in one set of cells (a tissue).

Finally, groups of similar genes often have overlapping functions in an organism, this is especially prevalent in all animals with a backbone, which includes human. This complicates studying genes as more than one needs to blocked before to the full repertoire of gene functions can become clear.

There is now a new technology available, named CRISPR interference, which can be used a to turn one or multiple genes on or off in just the tissue of interest. In the zebrafish in particular, these tools can be injected into the fertilised egg and this means that it will allow us to turn any gene of choice on or off in any cell or tissue in a complete animal. Injections can be done very efficiently and therefore testing of tens to hundreds of genes for a role in a particular disease or biological context is achievable. The zebrafish has become one of the workhorses for biological and biomedical research in recent years and we propose to develop CRISPRi technology as a resource in zebrafish that will benefit a large group of scientists that are currently using, or are thinking of adopting this model for their research.

We will make genetically modified zebrafish which are needed for this technique, and share these with colleagues around the country. We will refine the design of the tools and make sure these are as efficient as they can be. Finally we will combine the two, performing screens of collections of genes. This will identify new gene functions and provide a step-change in our understanding of a range of biological processes.

This technology will allow us to identify the genes which cause disease or control important biological processes much faster than previously possible.

Genetic screens have revolutionised our understanding of biology and disease, yet existing approaches to systematically test gene function have substantial limitations, especially when this is required in vertebrate organisms. The recent discovery of CRISPRi provides an exceptional opportunity to identify tissue-specific and redundant gene functions in vertebrates.

We will pursue this novel genetic approach in the zebrafish - a model organism that lends itself particularly well to large-scale approaches and in which we have world-leading expertise. In addition, the zebrafish affords unparalleled opportunities for the real time visualisation of cells and tissues.
CRISPRi uses enzymatically-inactive Cas9 (dCas9) fused to repression/activation domains to bind DNA and modulate transcription of target genes from endogenous promoter-enhancers using injected guide-RNAs. CRISPRi is non-toxic, rapid, cheap and scalable.

We will:
1. Generate and share dCas9 transgenics for specific cell types - screens will be performed by injection of gRNAs into these lines.
2. Design gRNAs against approximately 1000 genes using Agilent arrayed-library technology, genes will be chosen from UK community requests or encompass classes of high interest (eg. GPCRs, mechanoreceptors), maximising impact.
3. Perform screens against multiple assays and facilitate screens by shipping transgenics and libraries.

The ability to rapidly and cheaply modulate gene expression in a range of tissues will drive huge leaps in our understanding of biology, and is perfectly timed to exploit the wealth of human genetic data arising from a variety high throughput approaches.

Academic impact
The ability to modulate gene function rapidly and efficiently will have a wide impact in the zebrafish community and beyond. This research will establish new insights into a range of biologically important pathways and identify new pathways, which might contain suitable targets for drug discovery programmes. New technological advances will have impacts across a range of fields where genome editing is required. The integration of large scale CRISPR testing, validation and innovative computational analysis will lead to advances in integration of biology and computational biology, with potential impacts across many academic disciplines. The discoveries from this project will therefore significantly enhance the knowledge economy with new scientific advancement, as described in "academic beneficiaries".
Importantly, we are maximising impact by tailoring our transgenic lines and guides directly to requests from the UK scientific community and by establishing a website to disseminate the outputs of this programme.

Training impact
Training of our PDRA and Technician in state of the art gene manipulation will enhance the employee skill-base in the UK. In addition, the interface of computational biology and iterative design and validation of CRISPRs will equip the PIs and PDRA/Tech with important interdisciplinary mindsets and skillsets. This multidisciplinary group is an optimal environment for the generation of new researchers with transferable, in-demand skillsets.

Economic and competitive impact.
The success of this resource will strengthen competitiveness of the UK as a hub for new genome technologies. This is likely to attract new investment in the UK, for example in the form of european/international academic or industrial funding. In the longer term, our screens will increase our understanding of the genetic control of a range of biological processes, which may in turn identify clinical targets. This could lead to opportunities to engage industrial (Pharma/biotech) partners in developing new therapies.

Contribution to the public understanding of science
The strong visual appeal of the zebrafish embryo is an important and innovative way to promote bioscience to the general public, and school children in particular. Our extensive outreach activities will lead to a more informed and balanced view by the public of bioscience and the use of animals in biological research.

3Rs impact
We are enthusiastic advocates for applying the 3Rs (Replacement, Refinement, Reduction) to animal research. In the last decade, zebrafish use has become widespread, in some cases as a replacement for mice due to its lower neurophysiological sensitivity. Effective CRISPRi will enhance the appeal of the fish model and encourage a move away from rodent models. We plan to perform screens for labs not using zebrafish, further promoting its adoption.