First CRISPR single-nucleotide edited transgenic mice

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 3 billion nucleotides of four types: Adenine (A), cytosine (C), guanine (G), and thymine (T). 

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.

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, which is able to substitute one nucleotide into another. In this way, no DNA deletion occurs, but just one nucleotide substitution.

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.