Cystic fibrosis, sickle cell anemia, Huntington's disease and phenylketonuria are all examples of disorders caused by the mutation of a single nucleotide, a building block of DNA. The human DNA consists of approximately 6 billion nucleotides of four types: Adenine (A), cytosine (C), guanine (G), and thymine (T). In some cases, the difference of just one nucleotide can bring serious consequences. Scientists hope to cure these diseases by substituting the incorrect nucleotide with the correct one. However, it is technically challenging to replace a single nucleotide with the current gene editing tool, CRISPR-Cas9. Scientists at the Center for Genome Engineering, within the Institute for Basic Science (IBS) have used a variation of the popular gene editing technique CRISPR-Cas9 to produce mice with a single nucleotide difference. Their findings are published in Nature Biotechnology.

The most recent and highly successful CRISPR-Cas9 technique works by cutting around the faulty nucleotide in both strands of the DNA and cuts out a small part of DNA. Conversely, IBS biologists used a variation of the Cas9 protein (nickase Cas9, nCas9) fused with a protein called cytidine deaminase, a.k.a. Base Editor, which is able to substitute one nucleotide into another. In this way, no DNA deletion occurs, but just one nucleotide substitution. These types of deaminases have been developed and tested in cultured cell lines by David Liu's group at Harvard and Keiji Nishda and his colleagues at Kobe University in 2016. The IBS team advanced the technique further by applying it to mouse embryos.

▲ Difference between the traditional CRISPR-Cas9 technique and the CRISPR-nCas9-cytidine deaminase fusion in targeting the DNA position shown with the red C (cytosine). (Top) In the traditional CRISPR-Cas9 technique a guide RNA binds to the target RNA and the Cas9 protein (shown in blue) cuts both filaments of the DNA. This cuts out a small part of DNA (the part of the DNA shown in red becomes a bit shorter). (Bottom) A different version of Cas9 (nCas9, blue) is fused with the protein cytidine deaminase. This Cas9 cuts only one filament of the DNA and the cytidine deaminase modifies one nucleotide (from cytosine to thymine, T), producing a DNA of the same length with only one nucleotide difference.

The scientists tested the CRISPR-nCas9-cytidine deaminase fusion in mice by changing a single nucleotide in the dystrophin gene (Dmd) or the tyrosinase gene (Tyr). They were successful in both cases: Embryos with the single nucleotide mutation in the Dmd gene led to mice producing no dystrophin protein in their muscles, and mice with the Tyr mutation showed albino traits. Dystrophin is indeed connected with the muscular dystrophin disease and tyrosinase controls the production of melanine.

▲ Schematic layout showing how IBS scientists delivered the CRISPR-nCas9-cytidine deaminase fusion complex into mouse embryos to edit the gene dystrophin (Dmd) and tyrosinase (Tyr) and which results they obtained. The CRISPR-nCas9-cytidine deaminase fusion complex causes a single nucleotide substitution that leads to unfunctional proteins, which shows as reduction of dystrophin production in the muscles, in the case of Dmd, and albino features in the case of Tyr. The research team used two types of delivery methods: Microinjection of mRNA encoding for the fusion complex, or another technique called electroporation that opens pores on the membrane of the embryo allowing the preassembled fusion complex to enter inside the embryo. Then these embryos were transplanted into surrogate mothers. The experiment was successful in both cases. The research team obtained first generation and second generation mice bearing the mutation in the Dmd gene, as well as two albino pups out of seven, in the case of Tyr.

Moreover, these single-nucleotide substitutions appeared only in the target position. This is important because it means that only the correct nucleotide is substituted. "We showed here for the first time that programmable deaminases efficiently induced base substitutions in animal embryos, producing mutant mice with disease phenotypes. This is a proof-of-principle experiment. The next goal is to correct a genetic defect in animals. Ultimately, this technique may allow gene correction in human embryos," expressed KIM Jin-Soo, Director of the Center and leading author of this study.

Letizia Diamante