October 25, 2017

expert reaction to genome editing study on new class of base editors

A new study, published in Nature, presents a new class of ‘base editors’ – programmable protein machines that rearrange the atoms of one DNA to resemble a different base in the genome of living cells – that make it possible to individually replace all four bases of DNA selectively and efficiently, without causing any double-stranded DNA breaks.

Dr Helen O’Neill, Programme Director, Reproductive Science and Women’s Health, UCL, said:

“Base editing is the targeted and permanent alteration of individual subunits of our DNA.  DNA is made up of four bases: Adenine, Thymine, Guanine, Cytosine (A, T, G, C) which have specific pairing capacities; A binds to T and G binds to C.  Single base-pair changes can have subtle to severe effects on the performance or function of a gene.  Many disorders are the result of just a single base-pair error in an individual’s genome.  Current genome editing technologies are limited, firstly, in that they require the double-stranded DNA to be cut to allow repair, and secondly that they induce innate cellular repair mechanisms which can result in other unwanted base-pair insertions or deletions (known as indels) occurring.

“This paper has carried out a systematic multi-step (‘multi evolution’) strategy to develop and enable specific base editing of genomic DNA without the need for cutting DNA.  Recent base editing allowed only the conversion from Cytosine/Guanine to Thymine/Adenine, which meant its applicability was limited.  This paper has extended the capabilities of base editing to allow the conversion between Adenine/Thymine to Cytosine/Guanine.

“Using precise engineering, the authors meticulously analysed the architecture and components of enzymes and inhibitors to create these Adenine Base Editors (ABEs).  To do this, they examined the mechanisms of DNA adenine deaminases which are found in most organisms ranging from bacteria to humans.  These enzymes, which are necessary to allow for the conversion of adenosine to inosine, (which can be processed as guanine), work in different capacities in different organisms.

“The research presented here aimed to harvest the most precise version of each component to improve the specificity of the editing while simultaneously testing for broad usability (to enable these base editors to be used in many cell types and genes).  The authors note that precise editing windows can vary depending on the target.  The method was tested in human cells for different lengths of times.  They first aimed to make base changes at five different loci and compared this method (ABE) to the current-best method for genome editing (CRISPR/Cas9).  The ability to target and make base changes ranged from 10% – 68% (from 48 hours to 120 hours) using this method of adenine base editing (ABE) compared with only 0.47% – 4.22% using CRISPR/Cas9.  More importantly, the rate of unwanted indel formation was >0.1% using ABE compared to (up to) 10% using CRISPR/Cas9.

“They next tested the adenine base editing technique in human cells carrying mutations for two blood conditions, HPFH (hereditary persistence of fetal hemoglobin) and hemochromatosis (HHC).  These conditions are two of many disorders which result from single mutations where a Guanine is in the place of a Cytosine.  In both cell types, the bases were accurately converted with efficiencies ranging from 28-30%, showing no evidence of undesired mutations.

“The ability to now directly alter all four base-pairs with such specificity adds more ammunition to the genome editing artillery and will be incredibly powerful in the research of diseases and future restoration of disease-causing mutations.”

Dr Kosuke Yusa, Group Leader at the Wellcome Trust Sanger Institute, said:

“Although a high-fidelity Cas9 enzyme is under development, actual consequences of on-target double-stranded breaks are still uncontrollable and somewhat unpredictable.  Base editors give us more control on the outcome of on-target editing.  The new editor reported in this study represents substantial therapeutic potential to possibly correct disease mutations in the future.”

Prof. Robin Lovell-Badge, Group Leader, The Francis Crick Institute, said:

“This is both clever and important science. Base editing methods are powerful: through the use of CRISPR guide RNA to take a modified version of the Cas9 enzyme that lacks the ability to cut DNA but instead carries an enzyme that can convert one base (or letter in the DNA code) into another, they allow a specific base pair to be altered, in theory with high efficiency. Moreover, because it does not involve a double strand break in DNA, neither the non-homology end joining (NHEJ, which gives slightly unpredictable changes often involving small insertions or deletions – INDELs) nor homology directed repair (HDR, which requires a DNA template), are likely to interfere or confuse the outcome. This means that the ‘on-target’ changes are very predictable.

“However, the methods have been restricted to only making a C to T change in the code, with the partner in the base pair being changed from G to A. But this new work, which made use of extensive protein engineering, created a novel enzyme able to promote base editing from A to G, with the partner in the base pair being changed from T to C. Many genetic diseases are due to alterations (mutations) where a single base pair has been substituted for another. This makes these new base editing methods of great value in both basic research to make disease models and, in theory, to correct genetic disease – making either somatic (non-hereditable) or germline (hereditable) alterations. Much more research will be needed to show the methods are entirely safe and, perhaps, to find ways to increase their efficiency, which is already at an impressive 50%, but this is an exciting development.”

Prof. Darren Griffin, Professor of Genetics, University of Kent, said:

“The work represents another step change in the CRISPR story, now with a new technology that allows for a wider utility of base pair changes to be made. In a previous report, gene editing was only possible in a limited number of cases. The current technology allows for the change of many more of the DNA bases.

“Although this study was not of human embryos directly, they did correct a genetic defect in a human cell line and questions are bound to be asked about gene editing in an IVF setting. In essence however the wider implications and ethical arguments remain the same. As a society, we need to convince ourselves whether or not the benefits of gene editing in embryos outweigh the concerns. We also need to have an eye to the practicalities of putting gene editing into clinical practice. There are many practical hurdles that mean that the greatest benefit of the technology might well be as a research tool.”

* ‘Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage’ by Nicole M, Gaudelli et al. published in Nature on Wednesday 25 October.

Declared interests

Dr Kosuke Yusa: “No conflicting interests.”

Prof. Robin Lovell-Badge: “I have no conflicts of interest.”

None others received.