CRISPR is an extremely useful tool for mouse models but is not ready for use on human embryos

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By IMPC

Published 28th November 2018

A researcher in China claims to have produced the first genome-edited babies through using CRISPR. The IMPC uses the CRISPR/Cas9 system to generate the required knockout mouse lines. The project moved to use this technology because of its speed and accuracy, yet those involved in controlling and implementing CRISPR are keen to stress there is a long way to go before this technology should be implemented in human embryos. A recent blog post and video highlight how and why the IMPC uses this technique: Two new articles highlight the usefulness and intricacies of CRISPR/Cas9 technology & CRISPR-Cas9 and mouse genomics

Dr Lydia Teboul, Head of Molecular and Cellular Biology at the Mary Lyon Centre at MRC Harwell said “We have been using the CRISPR/Cas9 system over the past four years to create over 200 mouse lines with new mutations as part of our work for the International Mouse Phenotyping Consortium. We have learned a lot about the technology in the process. A key feature of these genome editing tools is the unpredictability of the outcome of applying them to embryos. Because of this, it is clear that the technology is not ready for use for assisted reproduction in clinic.”

Vchal/Shutterstock

The below article is republished under a Creative Commons license and highlights some of the main issues with altering human embryo DNA.


By Joyce Harper, UCL

A scientist in China claims to have produced the world’s first genome-edited babies by altering their DNA to increase their resistance to HIV. Aside from the lack of verifiable evidence for this non peer-reviewed claim, this research is premature, dangerous and irresponsible.

He Jiankui from the Southern University of Science and Technology in Shenzhen (which has reportedly since suspended him) said he edited the DNA of seven embryos being used for fertility treatment, so far resulting in the birth of one set of twin girls. He says he used the tool known as CRISPR to delete the embryos’ CCR5 gene (C-C motif chemokine receptor 5), mutations in which are linked to resistance to HIV infection.

If true, this is a significant advance in genetic science, but there are some very serious problems with this news. First, the research has not yet been published in a peer-reviewed journal so we cannot be sure of the exact details of what has been done. Instead, the scientist made the claims to the Associated Press news organisation, and the journalists involved haven’t been able to independently verify them. The parents of the allegedly gene-edited babies declined to be interviewed or identified.

Second, we know there can be significant problems with using existing gene-editing technology on human embryos. The main two issues are mosaicism, where the edited DNA does not appear in every cell of the embryo, and off-target effects, where other parts of the genome may also have been edited with unknown consequences.

Before genome editing becomes a clinical treatment, it is essential that scientists resolve both of these issues and eliminate other potential adverse effects on the embryo. We need comprehensive studies to show that genome editing is not going to cause harm to the future people it helps create. Any children born as a result of genome editing will also need long-term follow up. It would be vital to see the preliminary work that He has done to confirm that his technique has eliminated mosaicism and off-target effects, and it is surprising that he has not published this.

 

There is also a question over why gene-editing was used to tackle the particular issue of HIV transmission in this case. The reports suggest that the couples involved in the study were made up of HIV-positive men who had the infection under control and HIV-negative women. The risk of transmission of HIV for these couples would have been negligible, and there are well-established ways to prevent HIV transmission to the offspring of HIV-positive couples.

Finally, there is the wider ethical debate, which the scientist in this case has chosen to ignore. I was a member of the Nuffield Council on Bioethics working group. We spent 20 months examining all aspects of genome editing and published our report this summer. Our conclusion was that we needed a public debate before gene editing on embryos was carried out because this procedure takes reproduction to a new level.

Do we really need gene editing?

Most reports suggest that the potential main use of genome editing would be therapeutic genome editing to prevent the transmission of genetic diseases, such as cystic fibrosis. In most cases, couples at risk of transmitting a genetic disease to their children are able to prevent transmission using established techniques of screening before birth or even before an embryo is implanted via IVF. So perhaps editing embryos for therapeutic reasons is not the way forward.

But genome editing could also more controversially used for genetic enhancements, such as ensuring children have a particular desirable characteristic such as a certain eye colour. This raises even more ethical questions.

We also need legislation. In the UK, for example, the use of genome editing would be regulated by the Human Fertilisation and Embryology Authority, and would currently be illegal. Before this technique becomes a treatment, governments need to pass laws that will control and regulate it otherwise it could easily be misused.

With all this in mind, any research in this area needs to be peer-reviewed and published in the scientific literature, with all the necessary preliminary work, so that we can make a valued analysis of the technique. In bypassing this process, He has made our job much harder.The Conversation

Joyce Harper, Professor of Reproductive Science, UCL

This article is republished from The Conversation under a Creative Commons license. Read the original article.

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Eleonora Maino (ASHG poster: Wednesday 2-3pm, poster number 1107)

Pelizaeus Merzbacher Disorder (PMD) is a rare X-linked pediatric leukodystrophy, that affects approximately 1:100,000 children at birth. The disease is associated with severe motor and cognitive impairment and a limited life expectancy. PMD is caused by mutations in the PLP1 gene, encoding proteolipid protein 1, one of the main components of myelin. In healthy individuals, myelin forms an insulating layer around the nerve fibers allowing fast and efficient signal conduction in the nervous system. Mutations in the PLP1 gene interrupt this process, leading to the hallmark symptoms of PMD. While a variety of mutations can lead to PMD, the vast majority of cases are the result of duplications of the X chromosome region containing the PLP1 gene. Currently, there is no cure and treatment options are limited to symptom management, which fail to have any considerable impact on the quality of life or lifespan of PMD patients. Accordingly, there is an urgent need for the development of effective therapies for PMD patients.

Since the discovery of the novel genome editing technology CRISPR/Cas9, a variety of innovative strategies have been developed to correct genetic defects, including genome rearrangements such as duplication mutations. Here, we are implementing a CRISPR/Cas9-based approach to remove the Plp1 duplication and ameliorate disease manifestation in a PMD mouse model, with the eventual goal of providing a new treatment strategy not only for PMD, but for all genetic disorders caused by genomic duplications. To date, we have characterized a PMD mouse model containing a Plp1 duplication generated in the laboratory of Dr. Grace Hobson. This mouse model is an excellent model to test CRISPR/Cas9 strategies in vivo since it recapitulates both PMD human mutations and disease phenotypes.

To test the genome editing approach in vivo, we administered the CRISPR/Cas9 components via intracerebroventricular injection in the brain of newborn PMD pups, utilizing adeno-associated viral vectors 9 (AAV9) as a delivery vector. Preliminary analyses suggest that, 12 days after the injection, Plp1 expression both at the mRNA and protein level is reduced in PMD mice treated with CRISPR/Cas9 compared to GFP injected control mice. These data suggest that CRISPR/Cas9 promoted the removal of the Plp1 duplication in the treated mice. Next, we will optimize the treatment and assay for a potential attenuation of disease phenotypes.

Once completed, this project will provide the essential in vivo proof of concept to further develop the CRISPR/Cas9 system as a therapeutic option for PMD patients, opening a novel potential avenue for the treatment of genetic disorders caused by genomic duplications.

ASHG 2018 Annual Meeting – research using mouse models

Guest post by Robert Erickson (ASHG poster: Wed Oct 17th, 2:00pm – 3:00pm)

Human dominant, gain-of-function mutations in connexin 47 can cause lymphedema but are not always penetrant.  We sought to better understand the causes of variable penetrance and expressivity of one such mutant, R260C, by using CRISPR technology to create this mutant in mice of different genetic backgrounds.  Only mice homozygous for the mutation on the C57BL/6J genetic background showed a lymphatic phenotype.  As with other mouse models for human lymphedema-causing mutations, overt limb swelling was not seen.  Instead, there were increased numbers and size of lymph nodes, with increased lobulation.  In addition, there was abnormal chylous reflux and increased lymphatic branching in the ears.  Mice on genetic backgrounds which were 75% 129/J or A/J did not show abnormalities even when homozygous for the mutation.  These results suggest that modifying genes are important for the e expression of human CX47 gain-of-function mutations.

In a new study in cells, University of Illinois researchers have adapted CRISPR gene-editing technology to cause the cell’s internal machinery to skip over a small portion of a gene when transcribing it into a template for protein building. This gives researchers a way not only to eliminate a mutated gene sequence, but to influence how the gene is expressed and regulated.

Such targeted editing could one day be useful for treating genetic diseases caused by mutations in the genome, such as Duchenne’s muscular dystrophy, Huntington’s disease or some cancers.

CRISPR technologies typically turn off genes by breaking the DNA at the start of a targeted gene, inducing mutations when the DNA binds back together. This approach can cause problems, such as the DNA breaking in places other than the intended target and the broken DNA reattaching to different chromosomes.

The new CRISPR-SKIP technique, described in the journal Genome Biology, does not break the DNA strands but instead alters a single point in the targeted DNA sequence.

“Given the problems with traditional gene editing by breaking the DNA, we have to find ways of optimizing tools to accomplish gene modification. This is a good one because we can regulate a gene without breaking genomic DNA,” said Illinois bioengineering professor Pablo Perez-Pinera, who led the study with Illinois physics professor Jun Song.

In mammal cells, genes are broken up into segments called exons that are interspersed with regions of DNA that don’t appear to code for anything. When the cell’s machinery transcribes a gene into RNA to be translated into a protein, there are signals in the DNA sequence indicating which portions are exons and which are not part of the gene. The cell splices together the RNA transcribed from the coding portions to get one continuous RNA template that is used to make proteins.

CRISPR-SKIP alters a single base before the beginning of an exon, causing the cell to read it as a non-coding portion.

“When the cell treats the exon as non-coding DNA, that exon is not included in mature RNA, effectively removing the corresponding amino acids from the protein,” said Michael Gapinske, a bioengineering graduate student and first author of the paper.

While skipping exons results in proteins that are missing a few amino acids, the resulting truncated proteins often retain partial or full activity – which may be enough to restore function in some genetic diseases, said Perez-Pinera, who also is a professor in the Carle Illinois College of Medicine.

There are other approaches to skipping exons or eliminating amino acids, but since they don’t permanently alter the DNA, they provide only a temporary benefit and require repeated administrations over the lifetime of the patient, the researchers said.

“By editing a single base in genomic DNA using CRISPR-SKIP, we can eliminate exons permanently and, therefore, achieve a long-lasting correction of the disease with a single treatment,” said Alan Luu, a physics graduate student and co-first author of the study. “The process is also reversible if we would need to turn an exon back on.”

The researchers tested the technique in multiple cell lines from mice and humans, both healthy and cancerous.

“We tested it in three different mammalian cell lines to demonstrate that it can be applied to different types of cells. We also demonstrated it in cancer cell lines because we wanted to show that we could target oncogenes,” Song said. “We haven’t used it in vivo; that will be the next step.”

They sequenced the DNA and RNA from the treated cells and found that the CRISPR-SKIP system could target specific bases and skip exons with high efficiency, and also demonstrated that differently targeted CRISPR-SKIPs can be combined to skip multiple exons in one gene if necessary. The researchers hope to test its efficiency in live animals – the first step toward assessing its therapeutic potential.

“In Duchenne’s muscular dystrophy, for example, just correcting 5 to 10 percent of the cells is enough to achieve a therapeutic benefit. With CRISPR-SKIP, we have seen modification rates of more than 20 to 30 percent in many of the cell lines we have studied,” Perez-Pinera said.

The group built a web tool allowing other researchers to search whether an exon could be targeted with the CRISPR-SKIP technique while minimizing chances of it binding to similar sites in the genome.

Since the researchers saw some mutations at off-target sites, they are working to make CRISPR-SKIP even more efficient and specific.

“Biology is complex. The human genome is more than three billion bases. So the chance of landing at a location that’s similar to the intended region is not negligible and is something to be aware of with any gene editing technique,” Song said. “The reason we spent so much time sequencing extensively to look for off-target mutations is that it could be a major barrier to medical applications. We hope that future improvements to gene editing technologies will increase the specificity of CRISPR-SKIP so we can begin to address some of the problems that have kept gene therapy from being widely applied in the clinic.”

Research article: CRISPR-SKIP: programmable gene splicing with single base editors

A technology designed to improve CRISPR-Cas9 gene editing in mosquitoes and other arthropods succeeds with a high degree of efficiency, while eliminating the need for difficult microinjection of genetic material, according to researchers.

These results could pave the way for scientists examining a wide range of arthropods — and even some vertebrates — to more easily manipulate gene expression for fundamental research and practical applications such as control of vector-borne diseases like Zika virus and malaria, elimination of agricultural insect pests, and potentially gene therapy for human and animal health.

CRISPR — Clustered Regularly Interspaced Short Palindromic Repeats — is a relatively new and revolutionary way to modify an organism’s genome by precisely delivering a DNA-cutting enzyme, Cas9, to a targeted region of DNA. The resulting mutation can delete or replace specific DNA pieces, thereby promoting or disabling certain traits.

Current approaches in arthropods rely on delivering the gene-editing Cas9 directly to eggs by embryonic microinjection, a difficult and inefficient process that works in only a small number of species, noted Jason Rasgon, professor of entomology and disease epidemiology, Penn State College of Agricultural Sciences.

“In addition, microinjection can damage the eggs, and it requires expensive equipment and training to implement,” he said. “These restrictions dramatically limit the use of CRISPR-Cas9 technology across diverse species.”

To address these limitations, Rasgon’s lab developed ReMOT Control — Receptor-Mediated Ovary Transduction of Cargo — a method the researchers say can deliver Cas9 cargo to a targeted portion of the genome by easy injection into the blood of female arthropods, where it can be introduced into the developing eggs via receptors in the ovary.

Rasgon explained that during ovary and egg maturation, mosquitoes and other arthropods synthesize yolk proteins, which are secreted into the blood and taken up into the ovaries. The team hypothesized that molecules derived from these yolk proteins could be fused to Cas9 cargo and delivered into the egg at levels necessary to achieve genome editing in the embryo, bypassing the need for embryonic microinjection.

In the process of testing this hypothesis in Aedes aegypti, a mosquito that can spread pathogens such as dengue, chikungunya, Zika, and yellow fever viruses, the team identified a peptide known as P2C, a ligand that is recognized by ovarian receptors and functions in five other mosquito species as well.

To visually show that P2C could achieve uptake in the ovary, the researchers injected the peptide, infused with green fluorescent protein, into mosquitoes. They subsequently found fluorescence in more than 98 percent of primary oocytes.

For gene-editing experiments, the scientists targeted a gene that, when knocked out, results in white eye color rather than dark, providing a visible phenotype to aid in screening. They found that P2C, when bonded with the Cas9 enzyme, was able to deliver the gene-editing cargo to the ovary, where the desired mutation was achieved at a high rate of efficiency, resulting in genetically modified offspring.

The results of the study, published recently in Nature Communications, show that compared to embryo injection, gene editing by ReMOT Control is efficient and technically much easier to accomplish, according to Rasgon.

“Whereas the microinjection apparatus can cost thousands of dollars and require extensive training to use, the equipment for ReMOT Control injections costs approximately $2, and the technique can be learned in less than an hour,” he said.

“The lower cost and ease of adult injections makes this method a substantial improvement over existing embryo-injection techniques, putting gene-editing capability into the reach of nonspecialist laboratories and potentially revolutionizing the broad application of functional arthropod genetics.”

Research article: Targeted delivery of CRISPR-Cas9 ribonucleoprotein into arthropod ovaries for heritable germline gene editing

Among the most significant scientific advances in recent years are the discovery and development of new ways to genetically modify living things using a fast and affordable technology called CRISPR. Now scientists at The University of Texas at Austin say they’ve identified an easy upgrade for the technology that would lead to more accurate gene editing with increased safety that could open the door for gene editing safe enough for use in humans.

The team of molecular biologists found conclusive evidence that Cas9, the most popular enzyme currently used in CRISPR gene editing and the first to be discovered, has less effectiveness and precision than one of the lesser-used CRISPR proteins, called Cas12a.

Because Cas9 is more likely to edit the wrong part of a plant’s or animal’s genome, disrupting healthy functions, the scientists make the case that switching to Cas12a would lead to safer and more effective gene editing in their study published in the journal Molecular Cell.

“The overall goal is to find the best enzyme that nature gave us and then make it better still, rather than taking the first one that was discovered through historical accident,” said Ilya Finkelstein, an assistant professor of molecular biosciences and a co-author of the study.

Scientists are already using CRISPR, a natural mechanism used by bacteria to defend against viruses, to learn more about human genes, genetically modify plants and animals and develop such science-fiction-inspired advances as pigs that contain a fat-fighting mouse gene, leading to leaner bacon. Many expect CRISPR to lead to new treatments for human diseases and crops that have higher yield or resist droughts and pests.

This illustration shows the protein cas12a bound to a DNA helix (red and white). Credit: T. Yamano, H. Nishimasu & J. Rybarski.

But the CRISPR systems found in nature sometimes target the wrong spot in a genome, which–applied to humans–could be disastrous, for example, failing to correct for a genetic disease and instead turning healthy cells into cancerous cells.

Some previous studies have hinted that Cas12a is choosier than Cas9, but the research before now was inconclusive. This latest study, the researchers say, closes the case by showing that Cas12a is a more precise gene-editing scalpel than Cas9 and explaining why.

The team, led by graduate student Isabel Strohkendl and professor Rick Russell, found that Cas12a is choosier because it binds like Velcro to a genomic target, whereas Cas9 binds to its target more like super glue. Each enzyme carries a short string of genetic code written in RNA that matches a target string of genetic code written in the DNA of a virus. When it bumps into some DNA, the enzyme starts trying to bind to it by forming base pairs–starting at one end and working its way along, testing to see how well each letter on one side (the DNA) matches the adjacent letter on the other side (the RNA).

For Cas9, each base pair sticks together tightly, like a dab of super glue. If the first few letters on each side match well, then Cas9 is already strongly bound to the DNA. In other words, Cas9 pays attention to the first seven or eight letters in the genomic target, but pays less attention as the process goes on, meaning it can easily overlook a mismatch later in the process that would lead it to edit the wrong part of the genome.

For Cas12a, it’s more like a Velcro strap. At each point along the way, the bonds are relatively weak. It takes a good match all along the strip for the two sides to hold together long enough to make an edit. That makes it much more likely that it will edit only the intended part of the genome.

“It makes the process of base-pair formation more reversible,” Russell said. “In other words, Cas12a does a better job of checking each base pair before moving on to the next one. After seven or eight letters, Cas9 stops checking, whereas Cas12a keeps on checking out to about 18 letters.”

The researchers said that Cas12a still isn’t perfect, but the study also suggests ways that Cas12a can be improved further, perhaps one day realizing the dream of creating a “precision scalpel,” an essentially error-proof gene-editing tool.

“On the whole, Cas12a is better, but there were some areas where Cas12a was still surprisingly blind to some mispairing between its RNA and the genomic target,” Finkelstein said. “So what our work does is show a clear path forward for improving Cas12a further.”

The researchers are currently using these insights in a follow-on project designed to engineer an improved Cas12a.

 

Research article: Kinetic Basis for DNA Target Specificity of CRISPR-Cas12a

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.

Research articles:

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.

As highlighted in a recent post, there are a variety of new applications for CRISPR technology. A study out this week describes another use for this revolutionary gene editing tool, and one that is getting a lot of media coverage. The application, known as a ‘gene drive’ has already been tested in insects, but this is the first attempt in mammals.

Gene drives allow chosen mutations to be passed on to the majority of offspring, and therefore desired alleles can quickly spread through a population. There are a variety of reasons this could be a useful tool. In particular, it is hoped it could be used to help control invasive and problematic species, such as rodents in vulnerable environments or mosquitoes in areas with malaria. Gene drive technology may also prove useful for creating desired mouse lines.

The research has been published in the preprint server bioRxiv:

 Super-Mendelian inheritance mediated by CRISPR/Cas9 in the female mouse germline

Abstract

A gene drive biases the transmission of a particular allele of a gene such that it is inherited at a greater frequency than by random assortment. Recently, a highly efficient gene drive was developed in insects, which leverages the sequence-targeted DNA cleavage activity of CRISPR/Cas9 and endogenous homology directed repair mechanisms to convert heterozygous genotypes to homozygosity. If implemented in laboratory rodents, this powerful system would enable the rapid assembly of genotypes that involve multiple genes (e.g., to model multigenic human diseases). Such complex genetic models are currently precluded by time, cost, and a requirement for a large number of animals to obtain a few individuals of the desired genotype. However, the efficiency of a CRISPR/Cas9 gene drive system in mammals has not yet been determined. Here, we utilize an active genetic ‘CopyCat’ element embedded in the mouse Tyrosinase gene to detect genotype conversions after Cas9 activity in the embryo and in the germline. Although Cas9 efficiently induces double strand DNA breaks in the early embryo and is therefore highly mutagenic, these breaks are not resolved by homology directed repair. However, when Cas9 expression is limited to the developing female germline, resulting double strand breaks are resolved by homology directed repair that copies the CopyCat allele from the donor to the receiver chromosome and leads to its super-Mendelian inheritance. These results demonstrate that the CRISPR/Cas9 gene drive mechanism can be implemented to simplify complex genetic crosses in laboratory mice and also contribute valuable data to the ongoing debate about applications to combat invasive rodent populations in island communities.

Abstract made available under a CC-BY-NC-ND 4.0 International license.

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.

Genes associated with fat metabolism can be a real asset for species like polar bears

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.

Impaired fertility is common in cheetahs both in captivity and in the wild

“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”.

Number of IMPC significant phenotypes for selected mammalian species (source: http://bit.ly/conservationarticle)

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 aims to produce and phenotype knockout mouse lines for 20,000 genes

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.

Brendan Doe talks about his work in using Cytoplasmic Microinjection to generate CRISPR mediated mutations.

(more…)

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By IMPC

Published 20th April 2018

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