A high throughput pipeline for manipulation of the zebrafish genome to facilitate fundamental biological research and molecular medicine.
Genome Editing uses one of several rapidly-developing technologies to modify specific sites of interest in the genome. In its simplest form, it creates a series of mutations at a precisely defined target site in a gene of interest. Targeting can eliminate or modify a gene on both chromosomes in a single diploid cell. More sophisticated variants can introduce precise modifications by homologous recombination, including tagging of proteins, insertion of DNA at precise loci (for example, loxP sites).
The two major current technologies are TALENs and CRISPR/Cas. In each case, the reagents target a specific double stranded DNA sequence in the genome and create a chromosomal double strand break that triggers non-homologous end joining (NHEJ) repair. At significant frequency, such repair makes errors, creating small (1-20 bp) deletions, insertions or duplications, which can be detected by a PCR-based method.
By simultaneously introducing single or double stranded DNA constructs with homology to the cut site, it is also possible to insert DNA sequences into the break, thereby creating a precise gene conversion of interest.
A major bottleneck in analysis of vertebrate gene function is the generation time of model species. Typically, a year is required to genetically modify a mouse including targeting vector production, homologous recombination in murine ES cells and breeding to homozygosity. If combinations of several altered genes are required, further generations of mice must be bred. Genome Editing provides a pathway to increase throughput and slash costs by reducing the number of generations required to generate specific genotypes.
Another advantage of Genome Editing is the ability to rapidly modify specific genes in precise ways through homologous recombination in the live animal and to genetically modify essentially any organism in which RNA can be introduced into germ line cells.
| Advantages | Disadvantages | |
|---|---|---|
| TALEN | 32-34 bp specificity Few off-targets Can be made to any target |
Time to make reagents Low mutation rate in some case |
| CRISPR | High mutation rate Fast guide RNA synthesis Can pre-check efficiency in-vitro Economical |
Lower 13-16 bp specificity may give significant off-target problems Restricted site availablilty (requires 4 precisely positioned G residues) |
We are seeking funding to expand our facility for the production of mouse and human genome editing tools and genome-modified cells lines.
The facility was created in 2011, thanks to a Wellcome Trust Technology Development Grant to Corinne Houart, Jon Clarke, Uwe Drescher, Robert Hindges and Simon M. Hughes.
The Genome Editing team, comprising Dr Clinton Monfries and Ms Oniz Suleyman, implemented successfully zinc finger nuclease, TALE nuclease and CRISPR/Cas technology to generate mutant zebrafish.
Over the past three years, we have successfully created over 100 genome editing tools, mutations in 47 zebrafish genes, comprising a total of 61 alleles. In 5 cases so far, we have succeeded in bi-allelic mutation in a single step, both with TALEN and CRISPR technology. Our studies have revealed how to enhance the rate of mosaicism in F0. We have also succeeded in introducing loxP sites into the zebrafish genome at selected loci using both TALEN- and CRISPR-mediated reagents. For informartion on the available tools and lines, check our database.
To date, we have also provided support and/or reagents for more than 20 labs in the UK and abroad.
Funded by a Charity (Wellcome Trust), the facility aims to develop this cutting-edge technology and provide a service to the KCL, UK and international community. All reagents and GM animals are generated under a Creative Commons Licence, with due regard being paid to the rights of project initiators to timely initial publication.
Please see our Conditions of Use Policy.
Researchers aiming to commercialize or otherwise restrict access to reagents or animals have to disclaim this prior to any use of our facility as this will only be possible under very specific and restricted conditions.
Dr Clinton Monfries 020 7848 6548
Ms Oniz Suleyman 020 7848 6548
4th floor North, New Hunt’s House, King’s College London, Guy’s Campus
New users should contact Prof Corinne Houart 020 7848 6409, or Prof Simon Hughes 020 7848 6445.
Funded by the Wellcome Trust
Webpage design by Clinton Monfries, Grant Wray & Corinne Houart
Webpage coding by Grant Wray
Based on the Skeleton template by Dave Gamache
