CRISPR base-editing linked to off-target effects
Dr Kimberley Bryon-Dodd, Progress Educational Trust
04 March 2019

[BioNews, London]

Two new studies suggest that a form of CRISPR base editing is more error-prone than previously thought. 

The results of the studies published in the journal Science – which examined base editing methods in mouse embryos and rice – could have implications for the therapeutic potential of this form of genome editing.

'The single-base gene-editing method was believed to be safe but the new testing tool showed the contrary, so we should make clinical standards as soon as possible to ensure the safety of gene-editing technique,' said Dr Yang Hui of the Shanghai Institutes for Biological Sciences at the Chinese Academy of Sciences, who led the mouse study. 

Both studies concluded that cytosine base editing was associated with a large number of off-target mutations. This form of editing changes the DNA bases cytosine to thymidine. However, both studies also found that a different form of base editing, which converts adenine to guanine, was not linked to wide off-target effects. 

Scientists in the mouse study developed a new method of measuring off-target effects called Genome-wide Off-Target analysis. The researchers edited one cell of a mouse embryo when it was at two-celled stage. They introduced a fluorescent marker so that they could distinguish edited cells from unedited cells. After allowing the cells to grow and divide, they were then able to examine the daughter cells of both the edited and non-edited cells and check for mutations. 

The adenine base editor was associated with almost no off-target effects, but the cytosine base editor changed around one nucleotide in 20 million. That is about 20 times above the mutation rate in ordinary cell division. Some of these mutations occurred in genome regions that are associated with cancer development leading to concern that use of this form of base editing could increase cancer risk. 

Professor David Liu, whose group pioneered cytosine base editing said of the mouse study: 'It's a clever, elegant method designed to boost the signal so that we can now detect and understand these rarer types of guide-independent, off-target events.'

The rice study analysed DNA in 77 plants that had been altered with different base editors, controls and untreated plants. The researchers found that cytosine base editing roughly doubled the background mutation rate.

CRISPR base-editing technologies had been widely believed to be a safer form of genome editing and several companies are already investigating the feasibility of using it as a therapy for inherited genetic mutations in humans. The results from these two papers highlight that cytosine base editing may not be as safe as believed. However, the mouse study used higher levels of base editor than is likely to be needed for therapy in a clinical context, and it is possible that using a lower level of base editor might reduce this risk, the authors note.

Commenting in Wired, Dr Steve Murray at the Jackson Laboratory in Bar Harbor, Maine, who is part of a consortium developing safeguards and standards around therapeutic genome-editing, said: 'I don't think we or anybody can come up with a hard and fast rule, that above a certain number of errors it's not useful for anything and below which it's useful for everything. Every disease is different. Every therapy is different. Every patient is different.'


Chinese scientists find gene-editing tool highly likely to cause off-target mutations
CGTN |  1 March 2019
CRISPR base editing, known for precision, hits a snag with off-target mutations
STAT News |  28 February 2019
Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos
Science |  28 February 2019
Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice
Science |  28 February 2019
Gene editing is trickier than expected - but fixes are in sight
Wired |  28 February 2019
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Reproduced from BioNews with permission, a web- and email-based source of news, information and comment on assisted reproduction and human genetics, published by Progress Educational Trust.

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