Precise Gene Editing One SNP At A Time

Researchers in South Korea have produced the first transgenic mice using a base editor protein fused to CRISPR-Cas9.

AsianScientist (Mar. 8, 2017) – Researchers from the Institute for Basic Science (IBS) in South Korea have modified the CRISPR-Cas9 gene editing system such that single nucleotide replacements can be made without introducing double stranded breaks. Their findings have been published in Nature Biotechnology.

Human DNA has approximately three 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. For example, cystic fibrosis, sickle cell anemia, Huntington’s disease and phenylketonuria are all disorders caused by the mutation of a single nucleotide.

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 popular CRISPR-Cas9 gene editing technique. Instead, researchers from IBS’ Center for Genome Engineering have used a variation of CRISPR-Cas9 to produce mice with a single nucleotide difference.

CRISPR-Cas9 works by cutting around the faulty nucleotide in both strands of the DNA and cutting out a small section. In the present study, researchers used a variation of the Cas9 protein (nickase Cas9, nCas9) fused with a protein called cytidine deaminase, or Base Editor, which is able to substitute one nucleotide into another. In this way, no DNA deletion occurs, but a single nucleotide substitution.

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 DNA 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. Credit: IBS.

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). Mutations in Dmd cause muscular dystrophy and while Tyr controls the production of melanine. The researchers 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.

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,” said Professor Kim Jin-Soo, Director of the Center and lead author of this study.

The article can be found at: Kim et al. (2017) Highly Efficient RNA-guided Base Editing in Mouse Embryos.


Source: Institute for Basic Science; Photo: Shutterstock.
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