D1.3 Mutations and gene editing

My talk from the National Academies of Science and Medicine, the Royal Society, and the Chinese Academy of Science International Summit on Human Gene Editing, December 2015

Nature Methods, 2011

Molecular Therapy - Nucleic Acids

Duchenne muscular dystrophy is a severe debilitating genetic disease caused by different mutations in the DMD gene leading to the absence of dystrophin protein under the sarcolemma. We used CRISPR-Cas9 prime editing technology for correction of the c.8713C>T mutation in the DMD gene and tested different variations of reverse transcription template (RTT) sequences. We increased by 3.8-fold the editing percentage of the target nucleotide located at +13. A modification of the protospacer adjacent motif sequence (located at +6) and a silent mutation (located at +9) were also simultaneously added to the target sequence modification. We observed significant differences in editing efficiency in interconversion of different nucleotides and the distance between the target, the nicking site, and the additional mutations. We achieved 22% modifications in myoblasts of a DMD patient, which led to dystrophin expression detected by western blot in the myotubes that they formed. RTT optimization permitted us to improve the prime editing of a point mutation located at +13 nucleotides from the nick site to restore dystrophin protein.

BMC Genomics, 2018

The reporting of the first draft of the human genome in 2000 brought with it much hope for the future in what was felt as a paradigm shift toward improved health outcomes. Indeed, we have now mapped the majority of variation across human populations with landmark projects such as 1000 Genomes; in cancer, we have catalogued mutations across the primary carcinomas; whilst, for other diseases, we have identified the genetic variants with strongest association. Despite this, we are still awaiting the genetic revolution in healthcare to materialise and translate itself into the health benefits for which we had hoped. A major problem we face relates to our underestimation of the complexity of the genome, and that of biological mechanisms, generally. Fixation on DNA sequence alone and a 'rigid' mode of thinking about the genome has meant that the folding and structure of the DNA molecule-and how these relate to regulation-have been underappreciated. Projects like ENCODE have additionally taught us that regulation at the level of RNA is just as important as that at the spatiotemporal level of chromatin. In this review, we chart the course of the major advances in the biomedical sciences in the era pre-and post the release of the first draft sequence of the human genome, taking a focus on technology and how its development has influenced these. We additionally focus on gene editing via CRISPR/Cas9 as a key technique, in particular its use in the context of complex biological mechanisms. Our aim is to shift the mode of thinking about the genome to that which encompasses a greater appreciation of the folding of the DNA molecule, DNA-RNA/protein interactions, and how these regulate expression and elaborate disease mechanisms. Through the composition of our work, we recognise that technological improvement is conducive to a greater understanding of biological processes and life within the cell. We believe we now have the technology at our disposal that permits a better understanding of disease mechanisms, achievable through integrative data analyses. Finally, only with greater understanding of disease mechanisms can techniques such as gene editing be faithfully conducted.

Journal of Student Research, 2021

Gene editing technologies like the CRISPR-Cas9 system have forever changed the way we analyze genetics. As we now have a way to alter genetics, we can now investigate not just how a biological mechanism works, but wonder what we could do to make it better. This paper will discuss how biotechnology and gene editing are integrated into life sciences and biology, where most applications are found. It will be divided into four sections, each addressing how gene editing technology has advanced a field in life science. Firstly, the focus will be on viral detection systems SHERLOCK (Specific High-sensitivity Enzymatic Reporter UnLOCKing) and DETECTR (DNA Endonuclease Targeting CRISPR Trans Reporter), and their importance to the current global pandemic as well as other applications. Then the attention will be diverted into the rapidly popularizing work on gene drives and attempts to drive the evolution of populations of species to benefit mankind. Next, we will discuss the link between biot...

Health Science Inquiry

Basic Research in Cardiology, 2021

For a long time, gene editing had been a scientific concept, which was limited to a few applications. With recent developments, following the discovery of TALEN zinc-finger endonucleases and in particular the CRISPR/Cas system, gene editing has become a technique applicable in most laboratories. The current gain-and loss-of function models in basic science are revolutionary as they allow unbiased screens of unprecedented depth and complexity and rapid development of transgenic animals. Modifications of CRISPR/Cas have been developed to precisely interrogate epigenetic regulation or to visualize DNA complexes. Moreover, gene editing as a clinical treatment option is rapidly developing with first trials on the way. This article reviews the most recent progress in the field, covering expert opinions gathered during joint conferences on genome editing of the German Cardiac Society (DGK) and the German Center for Cardiovascular Research (DZHK). Particularly focusing on the translational aspect and the combination of cellular and animal applications, the authors aim to provide direction for the development of the field and the most frequent applications with their problems.

Gene Therapy, 2007

Targeted gene repair or targeted gene alteration is a molecular strategy that aims to correct single base mutations responsible for genetic diseases. The concept involves using singlestranded DNA oligonucleotides to direct a nucleotide exchange reaction at the genomic site of the mutation. Investigators have made significant progress in elucidating the mechanism(s) by which the mutation is corrected and have begun to focus on several viable targets that show great potential for clinical application. During the past several years, the field has witnessed a phase transition as the focus has switched from purely basic science to a sustained translational mode. We highlight the important advances over the last two to three years, some of which have moved the technology closer to the clinic while some others have introduced new reasons for caution.

2019

TA-SWISS, Foundation for Technology Assessment and a centre for excellence of the Swiss Academies of Arts and Sciences, deals with the opportunities and risks of new technologies.

Cellular Therapy and Transplantation

Genome editing is a breakthrough technology which consists of the process of precise modifications introduction into the genome of any organism. This scientific breakthrough provides a powerful tool for the errors correction in the nucleotide sequence of DNA. The development of the genome editing has inverted the concept of an available target for the therapeutic correction. The opportunity of highly precise and safe entering of single-and double-stranded breaks into the human DNA followed by natural DNA repair mechanisms has changed current approaches of the gene therapy and opened up new horizons in the treatment of numerous diseases. Genome editing is being developed to treat not only monogenic diseases but also infectious diseases and cancer. In the current review, we discuss the therapeutic application of genome editing.