CRISPR Cytosine Base Editor Found to Make Significant Off-Target Edits

CRISPR Cytosine Base Editor Found to Make Significant Off-Target Edits

The gene editing tool CRISPR-Cas9 allows precise edits to be made to genes to eliminate mutations or introduce new genes. The technology certainly has potential to cure genetic diseases, improve crop yields, and correct genetic mutations. CRISPR-Cas9 has been shown to be effective in the lab and studies have been conducted on human patients in China which appear to have been effective, although it is perhaps too early to tell.

However, questions have been raised about the safety of CRISPR-Cas9. Some studies suggest that far from being incredibly precise and having no off-target effects, unwanted edits may occur, and they could have consequences worse than the disease which scientists hope to cure.

Using CRISPR-Cas9 to remove defective genes is thought to involve a greater risk as both strands are cut. A form of CRISPR that acts as a base editor which can change single base mutations was thought to be safer option as the DNA is not cut.

A team of researchers in China and California have conducted a study to test the effectiveness of CRISPR base editors using a technique called Genome-wide Off-target analysis by Two-cell embryo Injection or GOTI for short. The technique was used to test CRISPR-Cas9 and CRISPR base editors to determine how precisely edits are made and whether there are off-target effects. The team used mouse embryos for the study and injected two different CRISPR base editors and compared these to CRISPR-Cas9 and a control. A marker was used to determine which cells had been edited. This allowed the edited cells to be separated and analyzed.

CRISPR-Cas9 was used to make cuts to the DNA in one test, a CRISPR base editor was used to change the DNA base adenine (A) for guanine (G) in another, and the third test involved a CRISPR base editor to change cytosine (C) to thymine (T).

The test involving CRISPR-Cas9 showed there were no more mutations than in the control, which had not been edited. It was a similar story with the base editor that changed the base A to G, but the base editor that swapped C for G saw 20 times as many mutations than had occurred in the control. These additional unintentional edits were quite rare, occurring at a rate of one unwanted edit every 20 million bases. However, that adds up to an extra 283 base changes in each embryo.

The researchers suggest that the problems with the cytosine base editor may not be down to CRISPR and only show that the method used still requires some tweaks to prevent off target effects. Many of the unwanted changes occurred on single stranded DNA that was being actively copied when the base editor was introduced. Changes could therefore be made to limit these off-target effects, such as changing how tightly the base editor binds to DNA. By making the base editor bind less tightly, off-target effects could be limited.

“All this additional information and knowledge that we gain about how well [CRISPR base editors] work brings us a step closer to using them to their full potential,” said Lars Steinmetz, co-author of the study.

The mouse embryo study is detailed in the paper – Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos – which was published in the Journal Science in February. DOI: 10.1126/science.aav9973

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