Adenine base editor excels at fixing point mutations | October 30, 2017 Issue - Vol. 95 Issue 43 | Chemical & Engineering News
Volume 95 Issue 43 | p. 3 | News of The Week
Issue Date: October 30, 2017 | Web Date: October 25, 2017

Adenine base editor excels at fixing point mutations

Editor is more efficient than CRISPR at single-base changes and makes fewer unwanted alterations
Department: Science & Technology
News Channels: Biological SCENE
Keywords: genomics, base editing, base editor, CRISPR, DNA, gene
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An adenine base editor changes an A to I and nicks DNA to induce the cell to convert I to G and T to C. 3' and 5' are DNA termini.
Credit: Adapted from Nature
Reaction scheme shows mechanism adenine base editor uses to change AT to GC.
 
An adenine base editor changes an A to I and nicks DNA to induce the cell to convert I to G and T to C. 3' and 5' are DNA termini.
Credit: Adapted from Nature

Many diseases, such as sickle-cell anemia, are caused by single-base mutations in genomic DNA. Scientists have long searched for methods to correct such single-base mutations, with hopes of possibly developing therapies for these genetic diseases.

Last year David R. Liu of Harvard University and coworkers developed a technique called base editing that fixes some single-base mutations. The method changes a cytosine-guanine (CG) base pair to a thymine-adenine (TA) pair (Nature 2016, DOI: 10.1038/nature17946 and C&EN, April 25, 2016, page 5). A major missing capability was the ability to go in the opposite direction by transforming AT to GC. Liu’s group has now closed that circle by developing adenine base editing (Nature 2017, DOI: 10.1038/nature24644).

Base editing uses a component of the popular genome-editing technique CRISPR, but it has some advantages over the standard CRISPR method. CRISPR is ideal for inserting and deleting DNA sequences at targeted locations in a genome, but base editing has the edge for single-base changes.

That’s because base editing is significantly more efficient than standard CRISPR at making single base substitutions. Also, base editing makes far fewer unwanted base insertions and deletions, called indels, which could lead to side effects in therapeutic applications.

The fundamental mechanistic difference between the two methods is that standard CRISPR cuts double-stranded DNA straight through, which can lead to indels, whereas base editing modifies DNA strands directly without making such cuts.

Liu and coworkers developed last year’s base editor by combining three proteins: a cytidine deaminase, a natural enzyme that converts C to uridine (U); a mutated Cas9 CRISPR enzyme that doesn’t cut DNA but uses an associated guide RNA to target specific DNA sequences; and a protein that prevents reversion of U back to C. After the cytidine deaminase changes C to U, the base editor nicks the strand opposite the modification to induce cellular machinery to replace G with A and change U to T.

Labs around the world have used this type of base editing to correct or install point mutations in a wide variety of organisms, including bacteria, fungi, rice, wheat, corn, tomatoes, fish, and mice. A Chinese group used it in human embryos to repair a mutation that causes the blood disease β-thalassemia.

Converting AT to GC is a much more difficult task than going from CG to TA because no natural enzyme converts A to a base resembling G or changes T to a C-like base in DNA. Liu and coworkers solved that problem by using directed evolution and enzyme engineering to convert a bacterial adenosine deaminase that normally works in RNA into a deoxyadenosine deaminase that converts A to inosine (I) in DNA. The researchers use a conjugate of the deoxyadenosine deaminase with a catalytically impaired Cas9 to convert A to I at a target site and to nick the opposite strand. The cell’s machinery then completes the process, converting I to G and T to C.

Adenine base editing’s average efficiency at making single-base changes is 53%, about an order of magnitude better than standard CRISPR. The new method also makes many orders of magnitude fewer indels. Working in human cells, Liu and coworkers used adenine base editing to correct a point mutation that causes the iron-storage disorder hemochromatosis and to install mutations that protect against sickle cell anemia.

Adenine base editing “is a really exciting addition to the genome-engineering toolbox,” comments Feng Zhang of the Broad Institute of MIT and Harvard, whose group pioneered the use of CRISPR for mammalian genome editing. “This work, in combination with the Liu lab’s earlier work on base editors, gives scientists new ways to make single-base changes at the level of DNA.” Zhang’s group just developed the first technique for using CRISPR to edit RNA instead of DNA.

Base editors “will be useful to the scientific community to create genomes altered with exquisite precision for a variety of basic science studies,” says Peter A. Beal of the University of California, Davis. “There is also the potential to develop these enzymes as therapeutics for genetic diseases caused by specific mutations. However, beyond relatively easily targeted organs, genomic therapies like this will require significant advances in methods to deliver the large biological molecules involved to the diseased tissues.”

This article has been translated into Spanish by Divulgame.org and can be found here.

 
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