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DNA Misspelling Correction Method is Very Accurate 게시판 상세보기

DNA Misspelling Correction Method is Very Accurate

- IBS scientists prove that a gene editing technique used for substituting a single nucleotide in the genome is highly accurate -

Researchers at the Center for Genomic Engineering, within the Institute for Basic Science (IBS) proved the accuracy of a recently developed gene editing method. This works as DNA scissors designed to identify and substitute just one nucleotide among the 3 billion nucleotides of our genome. "It is the first time that the accuracy of this base editor has been verified at the whole genome level," explains KIM Jin-Soo, leading author of this study. Published in Nature Biotechnology, this validation will help to expand the use of this method in the sectors of agriculture, livestock, and medicine, e.g. for gene therapy.

Rapid progress in gene editing tools has caused a frenzy excitement in the biology community. The main protagonist of the current third-generation DNA scissors is CRISPR - a tool that is quicker and cheaper than its predecessors. By cutting out a small DNA sequence, CRISPR-Cas9 and CRISPR-Cpf1 are used to silence or reduce the expression of faulty genes. However, last year, a new base editor method that does not cause random DNA deletions and insertions, but instead replaces only one DNA base, attracted the biologists' attention. These types of gene corrections are critical as several diseases are caused by the misspelling of one of the four basic components of DNA; adenine (A), cytosine (C), guanine (G), and thymine (T). Single-nucleotide errors in DNA are referred to as point mutations. Examples of diseases caused by point mutations include: cystic fibrosis, sickle cell anemia, and color blindness.

Unlike the existing third-generation DNA scissors, the base editor method consists of a variation of CRISPR-Cas9 (nCas9, nickase) fused with another enzyme called cytosine deaminase, which replaces the DNA component C with T. The scissors are directed to the correct position on the DNA by a guide RNA. However, up to now, it was not known whether the base editor was working only in the area of the faulty gene or if it was unnecessarily substituting Cs in other areas (off-target).

Just one month after reporting the first successful base editing in animals in Nature Biotechnology to modify a single nucleotide in dystrophin and tyrosinase genes, the same team demonstrated the accuracy of this method at the genome scale.


▲ Figure 1: Comparison between the two gene scissors: the third-generation CRISPR-Cas9 technique and the base editor. In the original CRISPR-Cas9 technique (top), the guide RNA (green) binds to the target DNA and the cleavage enzyme Cas9 (scissors) cuts out a small DNA sequence (red). A modified version of Cas9 called nCas9 (bottom) is different as it cuts off only one strand of DNA and the cytidine deaminase (pink) transforms a single cytosine (C) into uracil (U). Uracil (U) is then converted to thymine (T) by DNA replication.

In order to identify the correctness of the gene editing for the entire genome, IBS researchers modified the error-checking technique, known as Digenome-seq, in order to adapt it to the base editor method. Digenome-seq was used and validated last year, when the team analyzed the accuracy of CRISPR-Cpf1 and Cas9. IBS researchers also improved the computer program (Digenome 2.0) to identify off-targets more comprehensively and compared different guide RNAs, to find the one that reduces malfunctions and increases specificity.

Using this technique, the team demonstrated correctness of the base editor technique and they found it to be even more accurate than the current third-generation CRISPR-Cas9. The base editing technique induced C-to-T conversions in 1-67 sites in the human genome, while CRISPR-Cas9 caused cleavages in 30-241 sites, meaning that the base editor is making less off-target changes. "Therefore, it is expected that these base editors will be used as widely as the popular CRISPR technology," enthuses KIM.


▲ Figure 2: Comparison between DNA scissors' accuracy in the entire genome: standard CRISPR-Cas9 vs base editor. The vertical black bars represent C-to-T substitutions made in the genome, by the base editor (blue) and the leading third-generation technique CRISPR-Cas9 (red). The inner circle (gray) is a control where no gene editing tools were used. The red arrow shows the targeted C-to-T substitution, while all the other bars are off-target conversions. Since the black bars seen in the red zone are markedly higher than in the blue zone, the result suggests that the base editor acts more accurately on the target position than CRISPR-Cas9. The data is based on a previously-validated technique called Digenome-seq

Letizia Diamante

Notes for editors

- References
Daesik Kim, Kayeong Lim, Sang-Tae Kim, Sun-heui Yoon, Kyoungmi Kim, Seuk-Min Ryu and Jin-Soo Kim. Genome-wide target specificities of CRISPR RNA-guided programmable deaminases. Nature Biotechnology. DOI: 10.1038/nbt.3852

- Media Contact
For further information or to request media assistance, please contact: Mr. Shi Bo Shim, Head of Department of Communications, Institute for Basic Science (+82-42-878-8189, sibo@ibs.re.kr); Ms. Carol Kim, Global Officer, Department of Communications, Institute for Basic Science (+82-42-878-8133, clitie620@ibs.re.kr); or Dr. Letizia Diamante, Science Writer and Visual Producer (+82-42-878-8260, letizia@ibs.re.kr).

- About the Institute for Basic Science (IBS)
IBS was founded in 2011 by the government of the Republic of Korea with the sole purpose of driving forward the development of basic science in South Korea. IBS has launched 28 research centers as of January 2017. There are nine physics, one mathematics, six chemistry, eight life science, one earth science and three interdisciplinary research centers.

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