First-time development of single-nucleotide transgenic mice using CRISPR

Cystic fibrosis, sickle cell anemia, Huntington's disease, and phenylketonuria are all diseases caused by mutations in a single nucleotide. As a constituent unit of DNA, nucleotides are classified into four different types: adenine (A), cytosine (C), guanine (G), and thymine (T). Human DNA consists of approximately 3 billion nucleotides. In some cases, only one nucleotide change can cause a serious disease. Scientists hope to replace this incorrect nucleotide with a correct nucleotide to cure these diseases. However, there are technical challenges in replacing a single nucleotide with the current gene editing tool CRISPR-Cas9. Now, in a new study, researchers from the Institute for Basic Science (IBS) Genomics Engineering Center have developed a variant of this popular genetic editing technology, CRISPR-Cas9. Nucleotide editing mice. The results of the study were published online February 27, 2017 in the journal Nature Biotechnology, entitled "Highly efficient RNA-guided base editing in mouse embryos."

As a fruitful gene editing technique that has emerged in recent years, the mechanism of action of CRISPR-Cas9 is to cleave near a mutated nucleotide in the DNA duplex and to cleave a small DNA sequence. Instead, IBS researchers used a variant of the Cas9 protein: nickase Cas9 (nickase Cas9, nCas9), while confusing Cas9 with a protein called cytidine deaminase (CD). The CRISPR-nCas9-CD is capable of replacing one nucleotide with another, and is therefore also referred to as the Base Editor. In 2016, the David Liu team at Harvard University and the Keiji Nishda team at Kobe University in Japan have developed this type of deaminase and tested it in cell lines cultured in vitro. IBS researchers have further promoted its development by using this technology in mouse embryos.

利用CRISPR首次培育出单核苷酸转基因小鼠

IBS researchers tested in mice whether CRISPR-nCas9-CD is capable of correcting a single nucleotide in the Dmd gene (encoding dystrophin) or the Tyr gene (encoding tyrosinase). They succeeded in both genes: mice developed from embryos with a single nucleotide mutation in the Dmd gene did not produce dystrophin in their muscle, whereas the Tyr gene produced a single nucleoside. Acid-mutated mice exhibited an albino trait. Anti-dystrophin is indeed associated with muscle muscular dystrophy, while tyrosinase controls melanin production.

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