![\"Jan](\"https:\/\/news.embl.de\/wp-content\/uploads\/2019\/07\/Korbel_web.jpg\")
<\/a>To what extent do mutations in cancer cells interact with one another? That\u2019s one of the questions that EMBL\u2019s Korbel group<\/a> is trying to answer using CRISPR. \u201cThis is the principle of epistasis. In other words, we want to know whether one mutation in cancer can affect another mutation and if there is synergy between them,\u201d says group leader Jan Korbel. This is done by using cell lines originating from the same donor, but with different gene mutations. By deactivating a gene using CRISPR, the scientists can observe how this affects other genes.<\/p>\n \u201cWe also reconstruct fairly large-scale rearrangements in the genome that we have previously predicted to have a functional effect,\u201d says Korbel. \u201cHere we use the gene scissors to break the genome and remake it. The attempt is to repeat what we have observed in a disease. We make these rearrangements and anticipate a functional effect on gene expression.\u201d<\/p>\n CRISPR is a very versatile tool and can be used in all types of organisms, from yeast to mammalian cells. EMBL\u2019s Steinmetz group<\/a> uses this flexibility to their advantage. \u201cWe do a fairly broad coverage of the CRISPR-Cas 9 landscape,\u201d says group leader Lars Steinmetz. \u201cWe use it to study natural variants, to do genetic screens, and we think about how it could be used more safely for therapeutic editing applications.\u201d<\/p>\n The Steinmetz group studies the impact of single-nucleotide polymorphisms (SNPs) on phenotypes \u2013 that is, an organism\u2019s observable characteristics. SNPs are cases where a single DNA base has been substituted for another \u2013 a T instead of a C, for example \u2013 at a specific location in the genome. SNPs can have biomedical effects, such as increasing susceptibility to certain diseases or affecting the body\u2019s response to treatment. To study the effects of SNPs on an organism, the Steinmetz group carries out large-scale experiments. \u201cCRISPR enables us to do that with high efficiency. We developed an approach where we can engineer 10,000 to 100,000 different SNP variants in genomes of yeast,\u201d says Steinmetz. One goal of the project is to improve the methodology of CRISPR editing by increasing the efficiency of DNA repair at the cut site. This makes it more probable to get the desired edit.<\/p>\n Another project in the Steinmetz group involves research on off-target edits \u2013 unwanted side-effects of CRISPR gene editing. At the stage when a mouse embryo consists of only two cells, they modify one of these cells using one of several different CRISPR editing techniques that are under investigation. The genomes of the cell populations derived from these two cells are then compared. This makes it possible to determine which mutations are natural background mutations and which are caused by off-target edits. The number of off-target effects reveals the accuracy of different editing methods and makes it possible for the researchers to start finding ways to improve them.<\/p>\n The fruit fly \u2013 Drosophila melanogaster \u2013 <\/em>remains a model organism of choice for biological research, and is widely used at EMBL. Alessandra Reversi, a research technician in the Ephrussi group<\/a>, provides gene-editing services in fruit flies, creating genetically modified strains for scientists across EMBL.<\/p>\n \u201cUsing CRISPR is the best strategy for genome editing,\u201d says Reversi. \u201cThe advantage of CRISPR is that you can now modify the Drosophila <\/em>genome with even single-nucleotide precision. Compared with previous strategies, editing the fruit fly genome with CRISPR is more precise, and much easier, faster and cheaper. With this technique, scientists no longer need to perform time-consuming screens in order to identify flies with the desired modifications.\u201d<\/p>\n Fruit flies are not the only organisms for which EMBL provides gene-editing services. EMBL Rome has a Gene Editing and Embryology Facility that uses CRISPR to provide the whole of EMBL with gene-editing services in mice.<\/p>\n A relatively unusual application of CRISPR is epigenetic editing, which is being carried out in EMBL Rome\u2019s Hackett group<\/a> by postdoc Cristina Policarpi and PhD student Valentina Carlini. Epigenetics refers to the chemical changes that affect gene expression without actually altering the DNA sequence. One key type of epigenetic change is DNA methylation \u2013 the addition to the DNA molecule of a chemical group containing one carbon atom and three hydrogen atoms. Other types of epigenetic change involve chemical modifications of histones \u2013 the proteins around which our DNA is wrapped.<\/p>\n The Cas9 proteins used in Policarpi and Carlini\u2019s research have been modified so that they are unable to cut DNA: instead, they recruit other proteins. These recruited proteins then alter the epigenome through DNA methylation or histone modifications. \u201cThe main goal of the project is to understand how all these epigenetic marks contribute towards gene expression and genome organisation,\u201d says Policarpi. \u201cWe add or remove epigenetic marks only in small parts of the genome and we don\u2019t touch anything else, so we\u2019re sure that we\u2019re studying only the contribution of those marks to gene expression and regulation.\u201d<\/p>\n In another project in the Hackett group, CRISPR is used to knock out single genes in a pool of mouse cells \u2013 a different gene in each cell, until the 20,000 genes in the mouse genome have been covered. This makes it possible to study the effects of the absence of any gene on a phenotype of interest. An experiment on such a large scale is only possible with a tool like CRISPR. Policarpi has to laugh: \u201cOtherwise you would spend your whole life and that of your children and grandchildren in the lab!\u201d<\/p>\n There are still problems to overcome in the use of CRISPR. \u201cThe main concerns are editing at high efficiency and the issue of on-target editing accuracy,\u201d says Steinmetz. \u201cYou have to worry about off-target effects: apart from what I wanted to achieve, what else did I do to the cell by manipulating the system and introducing a new enzyme? These concerns get really important when you want to go into any form of therapeutic application.\u201d<\/p>\n Political and public concerns focus mostly on germline gene editing with CRISPR, where genes are edited so early in an organism\u2019s development that the changes are copied into all future cells, including sperm and egg cells. This means the edited genes will be passed on to future generations. These concerns have spiked since the case in November 2018 in which Chinese researcher He Jiankui claimed to have edited the genomes of two human embryos, using CRISPR to introduce a gene that would increase HIV resistance. This action was widely condemned by the scientific community as an experiment on humans that could have potential off-target effects, while its benefits remain questionable.<\/p>\n \u201cThere\u2019s of course a huge ethical debate about whether one should do germline editing in humans,\u201d explains Korbel. \u201cMy standpoint and EMBL\u2019s standpoint is that this should not be done. Introducing changes into a human being before he or she is born creates a huge issue of consent, as these mutations would remain in the germline for future generations, with consequences that are not clear. My position would be to have a moratorium for some years to come, to better understand the rates of errors that are introduced during gene editing with CRISPR, and to have a full ethical discussion.\u201d<\/p>\n Ewan Birney, director of EMBL\u2019s European Bioinformatics Institute (EMBL-EBI), agrees that this type of science should be closely regulated<\/a>. He points out that, in many countries, couples who carry a serious genetic disease and who are conceiving by in vitro\u00a0<\/em>fertilisation (IVF) can choose to implant only those embryos that do not carry the disease. This procedure is called pre-implantation genetic diagnosis, and is subject to strong national regulation. \u201cThe CRISPR approach would add an extra step of introducing CRISPR technology at the first-cell stage,\u201d Birney explains. \u201cThis would be followed by screening for a successful edit. Although this is technically possible, there are currently virtually no serious genetic diseases where pre-implantation diagnosis\u00a0would not\u00a0work but CRISPR\u00a0would.\u201d<\/p>\n Birney highlights that society must be able to trust in legislation to guarantee the ethical use of a new technology like CRISPR. \u201cIn the UK, the\u00a0Human Fertilisation and Embryology Authority\u00a0provides regulation enshrined in UK law in this area. Other countries in Europe have analogous legislation and regulation. This allows new technologies, such as mitochondrial donation [see pp. 50\u201351], to be developed in a way that is medically safe, scientifically and ethically sound, and supported by society.\u201d<\/p>\nStudying CRISPR\u2019s side-effects<\/strong><\/h2>\n
<\/a>Fruit flies for science<\/strong><\/h2>\n
<\/a>Editing the epigenome<\/strong><\/h2>\n
<\/a>What lies ahead<\/strong><\/h2>\n
Regulations and expectations<\/strong><\/h2>\n
<\/a>