̽��ֱ�� of Cambridge - Francis Crick Institute

Researchers call for greater awareness of unintended consequences of CRISPR gene editing

jg533 — Mon, 12 Apr 2021 08:11:52 +0000

CRISPR-Cas9 genome editing is a widely used research tool which allows scientists to remove and replace sections of DNA in cells, allowing them, for example, to study the function of a given gene or to repair mutations. Last year the researchers who developed CRISPR-Cas9 were awarded the Nobel Prize in Chemistry.

In the study published in the journal PNAS, scientists retrospectively analysed data from previous research in which they had studied the role of the OCT4 protein in human embryos during the first few days of development.

̽��ֱ��team found that while the majority of CRISPR-Cas9-induced mutations were small insertions or deletions, in approximately 16% of samples there were large unintended mutations that would have been missed by conventional methods to assess DNA changes.

Research is ongoing to understand the exact nature of the changes at the target sites, but this could include deletions of sections of DNA or more complex genomic rearrangements.

̽��ֱ��discovery highlights the need for researchers who use CRISPR-Cas9-mediated genome editing to edit human cells, whether somatic or germline, to be aware of and test for these potential unintended consequences. This is even more essential if they hope their work will be used clinically, as unintended genetic changes like this could lead to diseases like cancer.

“Other research teams have reported these types of unintended mutations in human stem cells, cancer cells and other cellular contexts, and now we’ve detected them in human embryos,” said Professor Kathy Niakan, group leader of the Human Embryo and Stem Cell Laboratory at the Francis Crick Institute and Professor of Reproductive Physiology at the ̽��ֱ�� of Cambridge, and senior author of the study.

“This work underscores the importance of testing for these unintended mutations to understand exactly what changes have happened in any human cell type.”

̽��ֱ��researchers have developed an open-source computational pipeline to identify whether CRISPR-Cas9 has caused unintended on-target mutations based on different types of next-generation sequencing data.

“We and others are trying to develop and refine the tools to assess these complex mutations, “ added Niakan.

“It is important to understand these events, how they arise and their frequency, so we can appreciate the current limitations of the technology and inform strategies to improve it in the future to minimise these mutations.”

Gregorio Alanis-Lobato, lead author and former postdoctoral training fellow in the Human Embryo and Stem Cell Laboratory at the Crick, said: “Conventional tests used to check the accuracy of CRISPR-Cas9 can miss the types of unintended on-target mutations we identified in this study. There’s still so much for us to learn about the effects of CRISPR-Cas9 technology and while this valuable tool is refined, we need to thoroughly examine all changes.”

There are important ongoing debates around the safety and ethics of using CRISPR-Cas9 genome editing on human embryos for reproductive purposes. And in 2019, there was international condemnation of the work of a researcher in China who edited embryos which led to the birth of twins. In the UK, its use on human embryos is closely regulated and is only allowed for research purposes. Research is restricted to the first 14 days of development and embryos are not allowed to be implanted into a womb.

̽��ֱ��data for this work related to embryos previously studied by the Crick’s Human Embryo and Stem Cell Laboratory. ̽��ֱ��embryos were at the blastocyst stage of early development, consisting of around 200 cells. They had been donated to research by people undergoing in vitro fertilisation (IVF) and were not needed during the course of their treatment.

̽��ֱ��research was led by scientists at the Francis Crick Institute, in collaboration with Professor Dagan Wells at the ̽��ֱ�� of Oxford. Kathy Niakan is Director of the ̽��ֱ�� of Cambridge’s Centre for Trophoblast Research, and Chair of the Cambridge Strategic Research Initiative in Reproduction.

Reference
Alanis-Lobato, G., et al: Frequent loss-of-heterozygosity in CRISPR-Cas9-edited early human embryos. PNAS, April 2021. DOI: 10.1073/pnas.2004832117

Adapted from a press release by the Francis Crick Institute.

CRISPR-Cas9 genome editing can lead to unintended mutations at the targeted section of DNA in early human embryos, researchers have revealed. This highlights the need for further research into the effects of CRISPR-Cas9 genome editing, especially when used to edit human DNA in laboratory research.

We and others are trying to develop and refine the tools to assess these complex mutations.

Kathy Niakan

Arek Socha on Pixabay

DNA

̽��ֱ��text in this work is licensed under a Creative Commons Attribution 4.0 International License. Images, including our videos, are Copyright © ̽��ֱ�� of Cambridge and licensors/contributors as identified. All rights reserved. We make our image and video content available in a number of ways – as here, on our main website under its Terms and conditions, and on a range of channels including social media that permit your use and sharing of our content under their respective Terms.

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Placenta is initiated first, as cells of a fertilised egg divide and specialise

jg533 — Wed, 23 Sep 2020 15:00:04 +0000

In a study published today in the journal Nature, researchers looked at the biological pathways active in human embryos during their first few days of development to understand how cells acquire different fates and functions within the early embryo.

They observed that shortly after fertilisation as cells start to divide, some cells start to stick together. This triggers a cascade of molecular events that initiate placental development. A subset of cells change shape, or ‘polarise’, and this drives the change into a placental progenitor cell - the precursor to a specialised placenta cell - that can be distinguished by differences in genes and proteins from other cells in the embryo.

“This study highlights the critical importance of the placenta for healthy human development,” said Dr Kathy Niakan, group leader of the Human Embryo and Stem Cell Laboratory at the Francis Crick Institute and Professor of Reproductive Physiology at the ̽��ֱ�� of Cambridge, and senior author of the study.

Niakan added: “If the molecular mechanism we discovered for this first cell decision in humans is not appropriately established, this will have significant negative consequences for the development of the embryo and its ability to successfully implant in the womb.”

̽��ֱ��team also examined the same developmental pathways in mouse and cow embryos. They found that while the mechanisms of later stages of development differ between species, the placental progenitor is still the first cell to differentiate.

“We’ve shown that one of the earliest cell decisions during development is widespread in mammals, and this will help form the basis of future developmental research. Next we must further interrogate these pathways to identify biomarkers and facilitate healthy placental development in people, and also cows or other domestic animals,” said Claudia Gerri, lead author of the study and postdoctoral training fellow in the Human Embryo and Stem Cell Laboratory at the Francis Crick Institute.

During IVF, one of the most significant predictors of an embryo implanting in the womb is the appearance of placental progenitor cells under the microscope. If researchers could identify better markers of placental health or find ways to improve it, this could make a difference for people struggling to conceive.

“Understanding the process of early human development in the womb could provide us with insights that may lead to improvements in IVF success rates in the future. It could also allow us to understand early placental dysfunctions that can pose a risk to human health later in pregnancy,” said Niakan.

̽��ֱ��research was led by scientists at the Francis Crick Institute, in collaboration with colleagues at the Royal Veterinary College, Vrije Universiteit Brussel, Université de Nantes and Bourn Hall Clinic. Kathy Niakan is incoming Director of the ̽��ֱ�� of Cambridge’s Centre for Trophoblast Research, and Chair of the Cambridge Strategic Research Initiative in Reproduction.

̽��ֱ��work was funded by the UK Medical Research Council, Wellcome and Cancer Research UK. It was approved by the UK Human Fertilisation and Embryology Authority (HFEA) and the Health Research Authority’s Research Ethics Committee.

Reference
Gerri, C. et al.: ‘Initiation of a conserved trophectoderm program in human, cow and mouse embryos.’ Nature, Sept 2020. DOI: 10.1038/s41586-020-2759-x.

Adapted from a press release by the Francis Crick Institute.

̽��ֱ��first stages of placental development take place days before the embryo starts to form in human pregnancies. This new finding highlights the importance of healthy placental development in pregnancy, and could lead to future improvements in fertility treatments such as IVF, and a better understanding of placental-related diseases in pregnancy.

Understanding the process of early human development in the womb could provide us with insights that may lead to improvements in IVF success rates in the future.

Kathy Niakan

Fluorescent images showing gene expression in human embryos at early and late stage of pre-implantation development, where blue is each cell of the embryo, green is a cell membrane marker, magenta is a placental gene expression.

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Global human genome study reveals our complex evolutionary history

jg533 — Thu, 19 Mar 2020 14:52:52 +0000

Uncovering a large amount of previously undescribed genetic variation, the study provides new insights into our evolutionary past, and highlights the complexity of the process through which our ancestors diversified, migrated and mixed throughout the world.

Published in the journal Science, the work involved the ̽��ֱ�� of Cambridge, the Wellcome Sanger Institute, the Francis Crick Institute and other collaborators. It is the first to apply the latest high-quality sequencing technology to such a large and diverse set of humans, covering 929 genomes from 54 geographically, linguistically and culturally diverse populations from across the globe.

̽��ֱ��results provide unprecedented detail of our genetic history, and highlights how it is characterised by multiple layers of complexity. Although genetic differences between populations reflect their diversity, many patterns are shared across continents, revealing both ancient and recent connections between populations.

Researchers in the ̽��ֱ�� of Cambridge’s Department of Genetics analysed the sequencing data to investigate evidence of interbreeding between the ancestors of modern humans and extinct human lineages such as Neanderthals and Denisovans, which occurred 40,000 to 60,000 years ago.

They found evidence that the Neanderthal ancestry of modern humans can be explained by just one major ‘mixing event’, most likely involving several Neanderthal individuals coming into contact with modern humans shortly after the latter had expanded out of Africa.

"Studying the patterns of Neanderthal ancestry in present-day humans hints at the structure of human communities more than 50,000 years ago. It is remarkable that patterns of Neanderthal ancestry are so similar in populations around the world today, and may have derived from a single Neanderthal population," said Dr Aylwyn Scally, a researcher in the ̽��ֱ�� of Cambridge’s Department of Genetics who was involved in the study.

In contrast, several different sets of DNA segments inherited from Denisovans were identified in people from Oceania and East Asia, suggesting at least two distinct mixing events. “This could suggest that multiple small groups of Denisovans once lived in different regions of Asia. We expect future discoveries of ancient DNA - perhaps from other extinct humans and perhaps even inside Africa - to tell us more about ancient population structure and diversity," said Dr Ruoyun Hui at the ̽��ֱ�� of Cambridge’s Department of Genetics, who also worked on the study.

Until recently, it was thought that only people outside sub-Saharan Africa had Neanderthal DNA. Now, the discovery of small amounts of Neanderthal DNA in west African people is most likely to reflect genetic backflow into Africa from Eurasia.

̽��ֱ��consensus view of human history is that the ancestors of present-day humans diverged from the ancestors of extinct Neanderthal and Denisovan groups around 500,000-700,000 years ago, before the emergence of ‘modern’ humans in Africa in the last few hundred thousand years.

Around 50,000-70,000 years ago, some humans expanded out of Africa and soon after mixed with archaic Eurasian groups. After that, populations grew rapidly, with extensive migration and mixture as many groups transitioned from hunter-gatherers to food producers over the last 10,000 years.

̽��ֱ��new data is freely available worldwide to benefit the study of human evolution and genetic diversity, including studies of genetic susceptibility to disease in different parts of the world.

̽��ֱ��team found millions of previously unknown DNA variations that are exclusive to one continental or major geographical region. Though most of these were rare, they included common variations in certain African and Oceanian populations that had not been identified by previous studies – variations that may influence the susceptibility of different populations to disease.

Medical genetics studies have so far predominantly been conducted in populations of European ancestry, meaning that any medical implications that these variants might have are not known. Identifying these novel variants represents a first step towards fully expanding the study of genomics to underrepresented populations.

However, no single DNA variation was found to be present in 100 per cent of genomes from any major geographical region while being absent from all other regions. This finding underlines that the majority of common genetic variation is found across the globe.

“ ̽��ֱ��detail provided by this study allows us to look deeper into human history, particularly inside Africa where less is currently known about the timescale of human evolution,” said Dr Anders Bergström, of the Francis Crick Institute and formerly the Wellcome Sanger Institute. “We find that the ancestors of present-day populations diversified through a gradual and complex process mostly during the last 250,000 years, with large amounts of gene flow between these early lineages. But we also see evidence that small parts of human ancestries trace back to groups that diversified much earlier than this.”

This study was funded by Wellcome and the Francis Crick Institute.

Reference
Bergström, A. et al. Insights into human genetic variation and population history from 929 diverse genomes, Science, March 2020; DOI: 10.1126/science.aay5012

Adapted from a press release by The Wellcome Sanger Institute.

A new study has provided the most comprehensive analysis of human genetic diversity to date, clarifying the genetic relationships between human populations around the world.

It is remarkable that patterns of Neanderthal ancestry are so similar in populations around the world today, and may have derived from a single Neanderthal population.

Aylwyn Scally

Matt Midgley

Handprints in the Cueva de las Manos, Patagonia, made by hunter-gatherers around 9,000 years ago

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Genome editing reveals role of gene important for human embryo development

sc604 — Wed, 20 Sep 2017 17:00:00 +0000

̽��ֱ��team used genome editing techniques to stop a key gene from producing a protein called OCT4, which normally becomes active in the first few days of human embryo development. After the egg is fertilised, it divides until at about 7 days it forms a ball of around 200 cells called the ‘blastocyst’. ̽��ֱ��study found that human embryos need OCT4 to correctly form a blastocyst.

“We were surprised to see just how crucial this gene is for human embryo development, but we need to continue our work to confirm its role” says Dr Norah Fogarty from the Francis Crick Institute, first author of the study. “Other research methods, including studies in mice, suggested a later and more focussed role for OCT4, so our results highlight the need for human embryo research.”

Dr Kathy Niakan from the Francis Crick Institute, who led the research adds, “One way to find out what a gene does in the developing embryo is to see what happens when it isn’t working. Now we have demonstrated an efficient way of doing this, we hope that other scientists will use it to find out the roles of other genes. If we knew the key genes that embryos need to develop successfully, we could improve IVF treatments and understand some causes of pregnancy failure. It will take many years to achieve such an understanding, our study is just the first step.”

̽��ֱ��research was published in Nature and led by scientists at the Francis Crick Institute, in collaboration with colleagues at Cambridge ̽��ֱ��, Oxford ̽��ֱ��, the Wellcome Trust Sanger Institute, Seoul National ̽��ֱ�� and Bourn Hall Clinic. It was chiefly funded by the UK Medical Research Council, Wellcome and Cancer Research.

̽��ֱ��team spent over a year optimising their techniques using mouse embryos and human embryonic stem cells before starting work on human embryos. To inactivate OCT4, they used an editing technique called CRISPR/Cas9 to change the DNA of 41 human embryos. After seven days, embryo development was stopped and the embryos were analysed.

̽��ֱ��embryos used in the study were donated by couples who had undergone IVF treatment, with frozen embryos remaining in storage; the majority were donated by couples who had completed their family, and wanted their surplus embryos to be used for research. ̽��ֱ��study was done under a research licence and strict regulatory oversight from the Human Fertilisation and Embryology Authority (HFEA), the UK Government's independent regulator overseeing infertility treatment and research.

As well as human embryo development, OCT4 is thought to be important in stem cell biology. ‘Pluripotent’ stem cells can become any other type of cell, and they can be derived from embryos or created from adult cells such as skin cells. Human embryonic stem cells are taken from a part of the developing embryo that has high levels of OCT4.

“We have the technology to create and use pluripotent stem cells, which is undoubtedly a fantastic achievement, but we still don’t understand exactly how these cells work,” explains Dr James Turner, co-author of the study from the Francis Crick Institute. “Learning more about how different genes cause cells to become and remain pluripotent will help us to produce and use stem cells more reliably.”

Sir Paul Nurse, Director of the Francis Crick Institute, says: “This is exciting and important research. ̽��ֱ��study has been carried out with full regulatory oversight and offers new knowledge of the biological processes at work in the first five or six days of a human embryo’s healthy development. Kathy Niakan and colleagues are providing new understanding of the genes responsible for a crucial change when groups of cells in the very early embryo first become organised and set on different paths of development. ̽��ֱ��processes at work in these embryonic cells will be of interest in many areas of stem cell biology and medicine.”

Dr. Kay Elder, study co-author from the Bourn Hall Clinic, says: "Successful IVF treatment is crucially dependent on culture systems that provide an optimal environment for healthy embryo development. Many embryos arrest in culture, or fail to continue developing after implantation; this research will significantly help treatment for infertile couples, by helping us to identify the factors that are essential for ensuring that human embryos can develop into healthy babies.”

Dr Ludovic Vallier, co-author on the study from the Wellcome Trust Sanger Institute and the Wellcome - MRC Cambridge Stem Cell Institute, said: “This study represents an important step in understanding human embryonic development. ̽��ֱ��acquisition of this knowledge will be essential to develop new treatments against developmental disorders and could also help understand adult diseases such as diabetes that may originate during the early stage of life. Thus, this research will open new fields of opportunity for basic and translational applications.”

Reference:
Norah M.E. Fogarty et al. 'Genome editing of OCT4 reveals distinct mechanisms of lineage specification in human and mouse embryos.' Nature (2017). DOI: 10.1038/nature24033.

Adapted from a Francis Crick Institute press release.

Researchers have used genome editing technology to reveal the role of a key gene in human embryos in the first few days of development. This is the first time that genome editing has been used to study gene function in human embryos, which could help scientists to better understand the biology of our early development.

This knowledge will be essential to develop new treatments against developmental disorders and could also help understand adult diseases such as diabetes that may originate during the early stage of life.

Ludovic Vallier

Dr Kathy Niakan/Nature

Day 2 embryo

̽��ֱ��text in this work is licensed under a Creative Commons Attribution 4.0 International License. For image use please see separate credits above.

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Social yeast cells prefer to work with close relatives to make our beer, bread & wine

Anonymous — Mon, 26 Oct 2015 13:05:30 +0000

̽��ֱ��findings, published today in the open access journal eLife, could lead to new biotechnological production systems based on metabolic cooperation. They could also be used to inhibit cell growth by blocking the exchange of metabolites between cells. This could be a new strategy to combat fungal pathogens or tumour cells.

“ ̽��ֱ��cell-cell cooperation we uncovered plays a significant role in allowing yeast to help us to produce our food, beer and wine,” says first author Kate Campbell.

“It may also be crucial for all eukaryotic life, including animals, plants and fungi.”

Yeast metabolism has been exploited for thousands of years by mankind for brewing and baking. Yeast metabolizes sugar and secretes a wide array of small molecules during their life cycle, from alcohols and carbon dioxide to antioxidants and amino acids. Although much research has shown yeast to be a robust metabolic work-horse, only more recently has it become clear that these single-cellular organisms assemble in communities, in which individual cells may play a specialised function.

For the new study funded by the Wellcome Trust and European Research Council, researchers at the ̽��ֱ�� of Cambridge and the Francis Crick Institute found cells to be highly efficient at exchanging some of their essential building blocks (amino acids and nucleobases, such as the A, T, G and C constituents of DNA) in what they call metabolic cooperation. However, they do not do so with every kind of yeast cell: they share nutrients with cells descendant from the same ancestor, but not with other cells from the same species when they originate from another community.

Using a synthetic biology approach, the team led by Dr Markus Ralser at the Department of Biochemistry started with a metabolically competent yeast mother cell, genetically manipulated so that its daughters progressively loose essential metabolic genes. They used it to grow a heterogeneous population of yeast with multiple generations, in which individual cells are deficient for various nutrients.

Campbell then tested whether cells lacking a metabolic gene can survive by sharing nutrients with their family members. When living within their community setting, these cells could continue to grow and survive. This meant that cells were being kept alive by neighbouring cells, which still had their metabolic activity intact, providing them with a much needed nutrient supply. Eventually, the colony established a composition where the majority of cells did help each other out. When cells of the same species but derived from another community were introduced, social interactions did not establish and the foreign cells died from starvation.

When the successful community was compared to other yeast strains, which had no metabolic deficiencies, the researchers found no pronounced differences in how both communities grew and produced biomass. This is implies that sharing was so efficient that any disadvantage was cancelled out.

̽��ֱ��implications of these results may therefore be substantial for industries in which yeast are used to produce biomolecules of interest. This includes biofuels, vaccines and food supplements. ̽��ֱ��research might also help to develop therapeutic strategies against pathogenic fungi, such as the yeast Candida albicans, which form cooperative communities to overcome our immune system.

Reference

Kate Campbell, Jakob Vowinckel, Michael Muelleder, Silke Malmsheimer, Nicola Lawrence, Enrica Calvani, Leonor Miller-Fleming, Mohammad T. Alam, Stefan Christen, Markus A. Keller, and Markus Ralser

Self-establishing communities enable cooperative metabolite exchange in a eukaryote eLife 2015, https://dx.doi.org/10.7554/eLife.09943

Baker’s yeast cells living together in communities help feed each other, but leave incomers from the same species to die from starvation, according to new research from the ̽��ֱ�� of Cambridge.

̽��ֱ��cell-cell cooperation we uncovered plays a significant role in allowing yeast to help us to produce our food, beer and wine

Kate Campbell

Metabolic cooperation in a social Baker’s yeast community. Pictured is a two-day old yeast community that grows as a colony. Different colours indicate cells producing and consuming different metabolites and nutrients.

̽��ֱ��text in this work is licensed under a Creative Commons Attribution 4.0 International License. For image use please see separate credits above.

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