Issue 85, April 2017
bullet CRISPR
bullet Innovation: CRISPR-Cas9 and the CRISPR-Cpf1 System
bullet CRISPRFlyDesign - Genome Engineering to Discover Conserved Biological Mechanisms
bullet Interview with Prof. Dr. Stefan Mundlos, Director of the Institute for Medical and Human Genetics at the Charité - Universitätsmedizin Berlin
bullet How Genome Scissors Help Identify Important Cancer Mutations
A new era of CRISPR experimentation is underway. CRISPR, the powerful, inexpensive and efficient genome editing technology allows for the most precise gene splicing. The CRISPR-Cas9 technology, which was developed in 2012, can be used to modify any organism's DNA and has the potential to create therapies for cancer, HIV, and genetic disorders. In addition, CRISPR can be used to create sturdier crops, malaria-resistant mosquitos as well as to combat skin bacteria and diarrheal infection. New CRISPR findings are constantly being revealed in major scientific journals, such as Science and Nature.

This cutting-edge technology has also raised legal, societal, and ethical issues. There are benefits and risks associated with human germline therapies and germline effects. According to a paper published by Leopoldina, the German National Academy of Sciences, the use of genome editing in research on human embryonic development is effective because there are significant differences in the embryogenesis of humans and animals. Yet, the paper rejects efforts to use genome editing for enhancement as this involves incalculable risks and raises fundamental ethical and social questions. Emmanuelle Charpentier, one of the inventors of CRISPR-Cas9 and the director of the Max Planck Institute for Infection Biology, encourages discussion between scientists, clinicians, ethicists, and the public to decide how to use this technology with ethical responsibility.

CRISPR experiments being carried out require a vast amount of time and money. Then again, Digital Trends predicts that it may only take several years until gene manipulation, designer babies, weaponized organisms, human augmentation, and pay-for-cure systems will be applied in the medical world.

The technology that uses CRISPR-Cas9 for gene-editing was invented in 2012 by the microbiologist, Emmanuelle Charpentier when she was at the Umeå Centre for Microbial Research, Sweden, and Jennifer Doudna from the University of California Berkeley. Their discovery is one of science's great success stories.

The three components involved in the process of CRISPR-Cas9 consist of two RNA molecules known as CRISPR-RNA and tracrRNA as well as the enzyme Cas9. Emmanuelle Charpentier's and Jennifer Doudna's laboratories re-engineered the two RNA molecules by combining them into one molecule. This turned CRISPR-Cas9 into a two-component system through which researchers could program one RNA molecule to target any complementary DNA sequence adjacent to a "GG"-motif for cleavage. The CRISPR-Cas9 enzyme scissors can modify any organisms' DNA and have the potential to create therapies for human diseases and genetic disorders.

In October 2015, Professor Charpentier became the director of the Max Planck Institute for Infection Biology in Berlin. She and her research team at the institute explore the regulatory processes in bacterial infectious diseases and they recently discovered a new mechanism known as CRISPR-Cpf1. The Cpf1 CRISPR-enzyme scissors are an elaboration of CRISPR-Cas9, cutting both RNA and DNA.  Cpf1 is smaller, naturally requiring only one RNA strand, targeting different sites, and opening up a wider range of applications. A paper published by Charpentier and her colleagues in Nature reveals that this CRISPR system may be even more useful for researchers than CRISPR-Cas9.

Emmanuelle Charpentier has won several prestigious awards, including a Leibniz Prize from the German Research Foundation in 2016. She has also founded two biotech companies that focus on further developing the CRISPR-Cas9 system.

The CRISPR/Cas9 system allows the manipulation of the genetic information of cells and organisms with unprecedented ease and precision. The technique has taken biomedical research by storm and is now an integral part of the research conducted in laboratories across the world. One of these is the CRISPRFlyDesign team in the Division of Signaling and Functional Genomics at the German Cancer Research Center (DKFZ) in Heidelberg. The goal of CRISPRFlyDesign is to develop CRISPR genome engineering tools for the popular model system Drosophila melanogaster, better known as the fruit fly.

The humble fruit fly has served as a workhorse for biologists for more than a century. Research on the fruit fly has uncovered several biological principles essential for the function of multicellular animals, including humans, ranging from the basis of inheritance to the identification of numerous genes implicated in common human diseases, such as cancer. Despite their highly divergent morphology and lifestyle, humans and flies are closely related in genetic terms. For example, 75% of known human disease genes are also found in flies and can be studied there. CRISPRFlyDesign therefore aims to enable the further molecular characterization of the fly genome.

To this end, the group has developed a suite of molecular tools, which are now in use in hundreds of research labs worldwide. These tools enable the targeted inactivation of genes in specific tissues or at certain times during development. To help scientists make the most of their genome engineering experiments, the team also maintains a CRISPRFlyDesign website, which provides detailed information about CRISPR experiments in the fly. Results from such experiments can then be followed up in other biological systems, including human tissue culture cells. Scientists from the Division of Signaling and Functional Genomics have also developed powerful bioinformatics tools, which can help researchers design and analyze such experiments.

Source & Image: German Cancer Research Center (DKFZ)


Prof. Dr. Stefan Mundlos is a human genetics expert. He is the director of the Institute for Medical and Human Genetics at the Charité - Universitätsmedizin Berlin, and the group leader of the Research Group Development & Disease at the Max Planck Institute for Molecular Genetics. Prof. Dr. Mundlos is the author of several publications, including his book, Limb Malformations: An Atlas of Genetic Disorders of Limb Development.  

Prof. Dr. Mundlos' research is based on genetic mechanisms of normal and abnormal development with a particular focus on the skeleton. Mechanisms of gene regulation and how they are influenced by genomic variation are an important aspect of his current work. He received the ESHG Award in 2016 for his work on the identification and characterization of disease genes and disease-causing mechanisms of gene regulation.

In this interview with the GCRI, Prof. Dr. Mundlos discusses the most exciting potential applications of CRISPR/Cas9. He describes ethical and safety issues associated with this genome editing technology, such as the concerns over genome editing in human embryos. He also highlights his current research on understanding how a certain class of mutations, known as structural variations, can affect the genome. To read the full interview, click here.
Source & Image: Max Planck Institute for Molecular Genetics
InnovationCECAD - Cluster of Excellence for Aging Research
The pace with which the CRISPR/Cas technology is changing biomedical research is astonishing. For the last 3 decades, mouse embryonic stem cells were the major tool for the recapitulation of human diseases in genetically modified mice. However, the generation of mutants using this technology is time consuming; it has been largely restricted to mice and does not allow the exclusive introduction of specific mutations, such as point mutations. Initially derived from a bacterial immune-defense system, CRISPR/Cas site-directed DNA endonucleases have, in contrast, enabled the fast and cost-effective generation of gene-targeted model organisms, allowing for the precise recapitulation of the genetic background in human diseases. Its simplicity in design, economic advantages, and exceptional efficiency have made the CRISPR/Cas9 system the method of choice for genetic editing in mice and other species.

Increasing life expectancy is concomitant with an increased risk of aging-associated diseases, including obesity, diabetes, atherosclerosis, cancer, and neurodegenerative diseases. These diseases pose enormous challenges for individuals and societies in terms of life quality and economic burden, thereby necessitating an urgent need for aging societies to address these health concerns. The Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases (CECAD) brings together researchers and clinicians at the University of Cologne with researchers at the new Max Planck Institute for Biology of Ageing in a unique research venture to advance understanding in this field. The mission of CECAD is to unravel the molecular mechanisms underlying lifespan regulation and aging-associated diseases to set the ground to achieve its long-term objective of developing novel therapeutic interventions.

The remarkable recent discovery that mutations in single genes can extend healthy lifespan implies that they can also reduce the impact of a broad spectrum of aging-related damage and pathology. To be able to effectively treat diseases, there must first be a thorough understanding of the mechanisms underlying them. Consequently, the CECAD transgenic core project provides access to state-of-the-art technology in model organisms and has implemented the CRISPR/Cas9 technology to precisely modify genes in fertilized one-cell mouse embryos. The technology enables the quick and effective validation of in vivo, the hypotheses derived from in vitro projects, as well as those from the analysis of primary patient material from clinical resources, paving the way for the development of preventive and novel therapeutic interventions. 
Source & Image: University of Cologne

BionaticHow Genome Scissors Help Identify Important Cancer Mutations
The CRISPR/Cas9 system, which is used for genome editing, is one of the fastest growing technologies with critical implications for biomedical research. As a programmable DNA scissor, the system allows for the precise cleavage at a predefined site in the genome of a cell. CRISPR/Cas9 can be used to destroy a gene by disrupting the reading frame of the sequence. This makes it possible to study the function of the gene in a cell. In recent work, this destructive power has been employed to advance cancer research.

Cancer arises in an organism when detrimental mutations accumulate in the genome. Improvements in DNA sequencing technologies have allowed for these mutations to be detected at an increasing speed and at a reduced cost in cancer patient material. However, in many cases it is not clear which of these mutations promote the tumor's growth and which mutations have no role during tumorigenesis. Cancer cells frequently harbor hundreds to thousands of mutations and the mutations can completely differ from patient to patient. Identifying the driver from the passenger mutations would definitely help in selecting the best treatment for the individual patient.

Indeed, the CRISPR/Cas9 system can be used to distinguish driver from passenger mutations. Because the molecular scissor can be programed to cut a specific sequence, the selective cleavage of the DNA that carries the cancer mutation can be achieved without affecting the wild type sequence. As a result, the cancer mutations can be rapidly inactivated in malignant cells and the consequence of this inactivation can be studied. Hence, this approach makes it possible to distinguish important from less important mutations and provides useful information on treatment options. The approach may also one day offer a novel route to a sophisticated cancer therapy if a cancer mutation-specific scissor cocktail can be administered to the cancer cells in the body. However, much research is still required to develop efficient and safe methods to deliver this scissor cocktail as a drug to patients.
Source & Image: Technische Universität Dresden