CRISPR gets crisper: Editing DNA at the single-base level
Life, for all its complexity, is built upon a relatively simple code, comprising only four letters – the chemical bases in the DNA molecule, adenine (A), guanine (G), cytosine (C) and thymine (T) – in intricate sequences many millions of letters long. Ever since the discovery of DNA, a holy grail for biologists has been the ability to re-write gene sequences to our own purposes. In recent years, the genome-editing technology known as CRISPR has made several leaps forward in this regard.
Off with the off-target effect
Already a number of distinct variations on the CRISPR technology exist, but the “standard” version works as follows: an enzyme called Cas9 is a molecular machine with the ability to bind to DNA in a highly specific, targeted manner, and chop the molecule. Artificially-introduced DNA strands can then fill the gap by a number of different methods. Since this involves cutting and then repairing DNA it may be considered a somewhat drastic method; the potential consequences of edits in the wrong place (off-target effects), or of failing to correctly repair a gap, can be severe. In a recent paper in Nature, Nicole Gaudelli, David Liu and colleagues at the Broad Institute of MIT and Harvard describe an adaptation of this technique which is able to make a directed modification to a single base without otherwise damaging the DNA.
Modifying just one base-pair out of the roughly 3 billion which make up a human genome may not seem like a big deal, but keep in mind: out of 50,000 known disease-causing mutations in humans, 32,000 are caused by just a single letter out of place, including such conditions as cystic fibrosis and beta thalassemia. So, the ability to edit DNA at the single base level, if made to work in living human tissue (and without unwanted off-target effects), would be of enormous medical significance.
The secret is not to cut
The new technique uses a modified version of the Cas9 enzyme which locates a specific place in the genome as usual; however, this particular variant has been stripped of its ability to cut DNA. Instead, an engineered enzyme has been attached which directly chemically modifies one DNA base. Distinct enzymes are needed for each specific base modification; a number of such enzymes have been previously reported on (e.g. 1, 2), but this particular paper discusses the development of a new class of adenine base editors (ABEs) which modify A-T base pairs to G-C (specifically, by removing an NH3 group from adenine to yield inosine, which is read as guanine in subsequent copying rounds). Since no naturally-occurring enzymes are known to exhibit this particular activity, the researchers employed an artificial evolution approach; by testing a huge range of variant enzymes in populations of E. coli bacteria under an antibiotic-based selection system, an effective candidate was found. The new Cas9-ABE system, when introduced into a number of human tissue-derived cell lines in the lab, was able to make the desired A – G modifications with around 30% efficiency.
This may seem low, but these are early tests of a new technology and, importantly, no evidence of off-target editing was seen. To be clear, this is still a long way from the actual application of this system in real, living humans. But it is a proof of concept which demonstrates at least the feasibility of precision genome editing as a tool against disease.
by Robin Floyd