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The use of the genome editing technique CRISPR/Cas9 in blind rodents is described by scientists publishing in the journal Nature with the authors reporting their introduction of a corrected gene to the eyes of the blind rats which partially improved vision.
All our previous output on this subject can be seen here. The SMC also produced a Factsheet on genome editing.
Prof. Robin Lovell-Badge, Group Leader, The Francis Crick Institute, said:
“This paper shows that it is possible to use a type of genome editing to introduce genes into a desired site within the genome even in non-dividing cells, such as neurons [nerve cells]. The method that many scientists are using to add or alter a gene in a precise way hijacks a normal cellular process of DNA repair called “homology directed repair or HDR”, and it relies on introducing a “DNA template” at the same time as the genome editing components, such as the CRISPR RNA guide and the Cas9 nuclease. The guide takes the nuclease to the right place in the genome, where it cuts the DNA. On repair via the HDR process, the template sequence is essentially copied into the genome. However, HDR does not operate in non-dividing cells, such as neurons, heart muscle, etc, making it a very inefficient mechanism to alter genes in these cells. The present authors show that it is possible to make use of the other major pathway of DNA repair, termed non-homology end joining (NHEJ), which simply sticks the ends of broken DNA back together. Usually this is imperfect and it is a good way to make mutations in genes. However, as shown in this paper, if a gene is added at the same time as the RNA guide and Cas9, then this will often get integrated at the site where the DNA was cut. The authors show that the components can be introduced in the same type of virus vector that is used in traditional gene therapy approaches. However, while the latter leads to integration of the exogenous gene anywhere in the genome, the addition of the genome editing components means that it will almost always integrate at the specific location chosen in advance. It is therefore possible to choose a “safe harbour” site, one that tolerates insertion of other genes without causing any problems and which helps to promote expression of the introduced gene. The authors go on to show how their methods can be used for gene therapy of a retina defect in rats, where they are able to target neutrons that do not divide.
“As reported, the methods are not super-efficient. For some genetic diseases it is not necessary to target all cells within the affected tissue, but levels of 5% or so would only give marginal benefit. However, with improvements in this type of technology, which seem inevitable these days, it is likely that the methods developed here could prove to be a very useful way of adding genes to non-diving cells, certainly for purposes of basic research, and perhaps eventually for gene therapy to treat otherwise incurable diseases.
“It is a complicated paper, and it does not quite reach the level as hyped in the press release, but it is indeed rather important.”
Dr Andrew Wood, Principal Investigator, MRC Human Genetics Unit, Institute of Genetics & Molecular Medicine, University of Edinburgh, said:
“This study is a really exciting development for therapeutic applications of genome editing. Although other groups have applied similar approaches in dividing cells grown in the laboratory, this is the first time that it has been used in non-dividing cells in a living animal.
“Before it can be applied to humans, it is now important to improve the efficiency with which the genome editing molecules can be delivered to the relevant cells.”
Dr Helen O’Neill, Embryology, IVF and Reproductive Genetics Group, Institute for Women’s Health, UCL, said:
“This is an elegant study which establishes new means for targeted integration of DNA in cells which are no longer dividing. These cells have long been considered a hurdle in somatic cell therapy. The major benefit is the ability to expand on current viral vector based gene therapy and combine this with improved Crispr/Cas9 delivery in non-dividing cells.
“Further work will need to be done on improving efficiencies, but this work certainly shows new avenues for alternative research into gene therapies.”
Prof. Robert MacLaren, Professor of Ophthalmology, University of Oxford, said:
“This is a significant advance. Retinitis pigmentosa (RP) is a genetic disease that causes blindness – it affects about 1 in 4,000 people in the UK. In this study the researchers used a bacterial protein to cut and repair defective DNA in a rat that has retinitis pigmentosa.
“The particular rat strain used is missing some of the genetic code in cells of the retina. The cells die off and the rat becomes blind – similar to patients with the same gene defect. The researchers use gene therapy to correct the DNA in the rat retinal cells, leaving them with some vision.
“This new technology of using bacterial proteins is called ‘CRISPR’ and it provides a way of correcting gene defects anywhere in the central nervous system. The CRISPR proteins were recently discovered to be a natural defence system that bacteria use to defend themselves against viruses by cutting viral DNA.
“Researchers are now using this mechanism to correct gene defects. Clinical trials are a long way off because the CRISPR proteins may cut DNA at other sites that may have untoward effects. Nevertheless, since ageing is defined as picking up DNA mutations, the ability to correct these mutations may in future provide us with a means of extending our lifespan as well as treating many diseases that relate to ageing.”
‘In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration’ by Keiichiro Suzuk et al. published in Nature on Wednesday 16th November.
Declared interests
Robin Lovell-Badge: Robin Lovell-Badge is employed at the Francis Crick Institute, and his lab carries out research using genome editing methods. However, he has no connection with any company using either this technology or with any gene therapy company. Indeed, his research is almost all at the level of basic research. He has no financial interest in the work described in the paper.
Dr Andrew Wood: No COIs
Prof. Robert MacLaren: I am a full time employee (Professor) at the University of Oxford and I have no relevant conflicts of interest. Specifically I have no commercial interests or grants relating to CRISPR technology and nor do I have any connections to the authors listed on the paper. I do see patients with retinitis pigmentosa and who might potentially benefit from this technology in future. I do not however see this as a conflict of interest.