CRISPR/Cas9 has revolutionised genetic research, with seemingly no end to its potential applications. Until recently genome engineering relied on the use of stem cells to target new mutations in mice but now researchers can perform genome editing directly in embryos or specific tissues. As with most projects that use gene editing technology, CRISPR/Cas9 has transformed the efficiency and enhanced the applications of the IMPC.
New CRISPR/Cas9-based techniques are constantly being developed, and existing systems adapted and improved, allowing increasingly sophisticated genetic changes to be made. Two papers published in the journal BMC Biology by Lanza, Gaspero, Lorenzo et al. (2018) and Codner, Mianné, Caulder et al. (2018) explore new advancements and highlight applications for these techniques.
The CRISPR/Cas9 system used in conjunction with single stranded DNA donors is revolutionising our ability to generate targeted mutations directly in the embryo. Whilst short synthetic DNA molecules facilitate this, the use of longer single-stranded DNA donors is a more recent addition to the genome editing toolbox. The two new articles summarised here compare long and short single-stranded donors in a high-throughput setting, both look at conditional knock-out mutants while also presenting advances for the generations of point mutations.
In the first study, led by researchers in Lydia Teboul’s group at the MRC Harwell institute, long single-stranded molecules are utilised to facilitate the generation of conditional alleles. They also apply the system to the introduction of point mutations remote from the recognition site of active Cas9/sgRNA complexes, which up to now has not been possible. This last technique is particularly valuable for human genomic sequencing since it enhances our ability to replicate human mutations in mice.
Alongside this breakthrough the researchers also highlight the unpredictability of this technique. As well as on-target integrations, the system can also produce an array of incorrect alleles. These include unintended point mutations, small or larger sequence rearrangements, and additional donor integrations. Such events are unpredictable by-products and therefore must be omitted in the process of validation of newly established mutant lines.
However, these by-products do not reduce the value of the new system. Instead, they illustrate the importance of a comprehensive validation of new mutants, including sequencing of the locus and copy counting of the number of copies of donor integrations. This process will likely become simplified over time, and issues overcome as new technological advancements are made. In the meantime, although unpredictable, the existing strategies remain efficient at generating desirable mutants.
Author on the paper Lydia Teboul said “An increasing body of evidence is being compiled to indicate that model validation is the newest challenge for the community. After all, the quality and reproducibility of research based on genome editing mutants depends entirely on the thorough characterisation of the mutant in question.”
The aim of the second study was to look at scaling production of conditional null alleles to create IMPC mouse lines. The research, led by Jason Heaney at Baylor College of Medicine, tested the feasibility of using CRISPR/Cas9 gene editing technology to generate conditional knockout mice using Cas9-initiated homology-driven repair (HDR), with both short oligonucleotides and longer single stranded DNA.
The results demonstrate that using pairs of short oligodeoxynucleotides can generate conditional null alleles at many loci; however, at scale there are inefficiencies with this process. On the other hand, long single stranded DNA donors may enable high-throughput production of conditional alleles. Although long single stranded DNA donors are most efficient at generating conditional alleles, pairs of short oligodeoxynucleotides are a viable alternative when use of a long single stranded donor is not feasible due to distance between loxP sites or complexity of sequence between loxP sites. Importantly, in agreement with the Teboul group, random integration of donor DNA and mutagenesis events at the target integration sites were detected when using either type of DNA donor.
Author on the article Jason Heaney said “Single stranded DNA donors are a critical component of the CRISPR/Cas9 genome editing toolbox and are an invaluable resource for producing conditional knockout alleles in mice. But, given the preponderance for random integration and potential for mutagenesis at sites of HDR, new mouse models produced with these donor DNAs must be carefully screened.”
Both studies highlight that it is essential to screen the sequence errors to check for point mutations, rearrangements and additional donor integrations. Researchers involved in genome editing face several challenges when using CRISPR/CAS9 technology and this latest research show it is essential to understand the unpredictability of different systems. Additionally, standards for the validation and documentation of mutants would be extremely beneficial to the field, and help to ensure quality and research reproducibility.
Codner GF, Mianné J, Caulder A et al. (2018) Application of long single-stranded DNA donors in genome editing: generation and validation of mouse mutants. BMC Biology 16: 70.
Lanza DG, Gaspero A, Lorenzo I et al. (2018) Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles. BMC Biology 16: 69.
The International Mouse Phenotyping Consortium (IMPC) has been predominantly interested in using mouse models to understand human health and disease. In a new study in the journal Conservation Genetics researchers have found another intriguing use of IMPC data.
By comparing genetic functional data from the IMPC with other non-human animals, it may be possible to identify genes relevant for the normal development in those species. For example, by comparing mouse genetic functional data with genomic data for selected species with specific diseases, improved breeding management could be implemented.
To test this potential application researchers at the European Bioinformatics Institute (EMBL-EBI) and Queen Mary University London (QMUL), alongside colleagues from the IMPC, compared genetic functional data from mice with genomic data from gorillas, showing how such analyses could aid in the identification of genes essential for healthy development.
As well as gorillas, the researchers highlighted other examples, including cheetahs, polar bears, wolves, pandas and cattle. This type of analysis could improve the current management approaches to breeding endangered species, by allowing researchers to identify the matches that are most likely to produce healthy offspring or select breeders to preserve genetic variation relevant for adaptation.
Heart disease is a common cause of death for gorillas in captivity and cheetahs suffer from impaired fertility both in captivity and in the wild. By identifying gorilla genes linked to heart disease or cheetah genes linked to infertility, researchers could help better understand the cause for the condition, which is the first step to envisage ways to prevent it. Similarly, this type of data could help identify genes linked to adaptation in certain mammals. For example, genes associated with fat metabolism can be a real asset for species like polar bears, which have diets rich in fats in the extreme environment of the Arctic.
“When the number of individuals of a species dramatically decreases, loss of genetic variation also takes place”, explains Violeta Muñoz-Fuentes, Biologist at EMBL-EBI. “This can result in many offspring not surviving, or exhibiting genetic defects linked to fertility or health problems.”
“Many zoos and wildlife conservation centres are seeing excellent results through their breeding programmes. Currently, many focus on minimising inbreeding. By adding a functional genetic dimension to the selective process, conservation geneticists can identify the crosses that would, for example, avoid a gene variant linked to disease in the offspring. It is nevertheless important to keep in mind that for a genetic rescue approach to be successful in the long term, the conditions that led to the decrease of individuals need to be removed; otherwise, the accumulation of deleterious alleles will likely take place again”.
Although this type of research is still in its early stages, gene functional knowledge is a powerful tool for maximising adaptive genetic diversity within a species and even for reducing genetic variants that negatively affect an individuals’ health and survival. With the accumulation of gene function annotation by the IMPC, as well as technical advancements in gene editing such as CRISPR/Cas9, the hope is that this method of comparing genome information between laboratory mice and endangered wildlife will help in future conservation projects.
The IMPC would like to encourage conservation geneticists, conservation centres and zoos to get in touch if they are interested in using IMPC data for conservation purposes.
Scientists have identified a network of genes that could play an important role in the development of metabolic diseases such as diabetes. A research team from the Helmholtz Center Munich and the International Mouse Phenotyping Consortium (IMPC) led the work that is published in ‘Nature Communications‘.
The development of metabolic diseases like diabetes is a complex process. As well as lifestyle and environmental factors, many different genes are responsible for the pathogenesis of both type 1 and type 2 diabetes. These genes encode information on how to assemble individual proteins that function in glucose metabolism.
Many genes that play an important role in the development of human diseases are still unknown. It is only by deciphering the causal genetic links that we can understand diseases, develop therapeutic interventions, or even prevent an outbreak. Thus, the new diabetes genes discovered in this study could be used, for example, as biomarkers for individual risk prediction, early diagnosis of the disease, or personalised approaches for treatment.
Twenty-three new candidate genes for diabetes in humans
As part of the IMPC, knockout mice – each lacking a specific gene – were examined for metabolic dysfunction. Using this method, researchers are trying to establish whether the missing gene is involved in important metabolic processes and can be linked to human diseases.
“Our analysis of the phenotyping data has identified a total of 974 genes whose loss has strong effects on glucose and lipid metabolism,” said Martin Hrabě de Angelis, who led the study and is the Chair of Experimental Genetics at the Technical University of Munich. “For more than a third of the genes no connection to metabolism was known previously.”
In addition, the researchers that teamed up with first author Dr. Jan Rozman, report that the functions of 51 of the discovered metabolic genes in the mouse were hitherto completely unknown. When compared with genome data collected in humans, they found that 23 of these also appear to play a role in human diabetes. “They are new candidate genes, and mice that lack these genes may be important models to investigate impaired glucose metabolism and diabetes,” explains Rozman, who coordinates the metabolic phenotyping at the German Mouse Clinic as part of the IMPC. One of these genes is C4orf22, which appears to be involved in insulin action in participants of the diabetes study “Tübingen Family Study (TÜF)”.
Interestingly, according to the bioinformatician and co-author Dr. Thomas Werner, these genes were also similar in their structure – many had common genetic elements. The scientists therefore assume that these genes belong to a network. In the future, they want to investigate these new regulatory structures and to explore to what extent they allow the prediction of gene functions of so-far unknown genes.
Rozman et al. Nature Communications 2018
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This study in Nature Genetics reveals hundreds of new insights into gene function and human disease. The paper describes the analysis of 3,328 genes by the IMPC, representing approximately 15% of the mouse genome. 360 new disease models were identified. Moreover, the team identified new candidate genes for diseases with unknown molecular mechanisms. More than half of the genes analysed have never been investigated in a mouse before, and, for 1,092 genes no molecular function or biological process were previously known.