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New gene-editing system can swap single DNA letters

by  /  25 May 2016
Trading bases: Researchers used a gene-editing system to open up the DNA coil, giving access to an enzyme that switches DNA letters.
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This article is more than five years old. Autism research - and science in general - is constantly evolving, so older articles may contain information or theories that have been reevaluated since their original publication date.

A new gene-editing system allows researchers to switch one DNA letter for another in cells. It may enable researchers to engineer animals to evaluate the consequences of these ‘point mutations.’

The new tool builds off of the CRISPR-CAS9 system, in which a scissor-like enzyme homes in on specific spots in the genome using a piece of customized RNA as a guide. The standard tool can make point mutations, but is inefficient, says lead researcher David Liu, professor of chemistry and chemical biology at Harvard University.

This inefficiency stems from CRISPR-CAS9’s mode of action, which is to sever both strands of DNA, causing the cell’s repair machinery to rapidly fix the break. In many cases, the repair machinery seals the break by inserting or deleting random DNA instead of creating the desired point mutation. Liu’s study appeared in April in Nature1.

In the new method, the researchers disabled the CRISPR system’s ability to cut through DNA, preventing the random insertions or deletions. Instead, they fused it with an RNA enzyme that snips out one chemical base and replaces it with another — specifically, replacing a cytosine (C) with a uracil (U).

DNA is made up of four chemical bases: adenine (A), guanine (G), cytosine and thymine (T). In RNA, uracil replaces thymine. In the double-stranded DNA molecule, an A always pairs with a T, and a C always pairs with a G.

The new enzyme switches a C-G pair to a U-G. The researchers made additional tweaks to prevent the cell from fixing this ‘mistake.’ They gave the new system the ability to nick the DNA strand that contains the G, causing the cell to ‘repair’ the mismatch by exchanging the G for an A. An A then pairs with a U. When the DNA is copied, the cell replaces the U with a T, creating a T-A pair in place of the original C-G combination.

Frankenstein fusion:

Researchers used the new method to make a similar change in the APOE4 protein. Studies suggest that a C-to-T flip in this protein lowers the risk of Alzheimer’s disease. The swap works as much as 75 percent of the time, researchers found.

The new system has few apparent off-target effects. It does alter any other cytosines that are within five base pairs of the target base, however. In the case of APOE4, the method swapped two other cytosines within this five-base pair stretch, but neither mutation changed the resulting protein sequence. Still, researchers must assess the tool’s effectiveness for any specific mutation on a case-by-case basis, Liu says.

The new method can make only two types of pair changes so far: C to T or G to A. But Liu and his team are looking for bacterial enzymes they could deploy in a similar Frankenstein-like fusion to generate other mutations. “In an ideal world, we would end up with a whole set of base editors that one could pull off the shelf depending on the type of change you wanted to make,” Liu says.


References:
  1. Komor A.C. et al. Nature 533, 420-424 (2016) PubMed
TAGS:   autism, CRISPR, technology