|Chang Xing and his team from Shanghai Health Institute reported new method of DNA base editting|
On October 10, international academic journal Nature - method (Nature the Methods), published online by the Chinese academy of sciences Shanghai life science institute/institute of Shanghai jiaotong university school of medicine, health science Chang Xing team entitled “Targeted AID - mediated mutagenesis (TAM) enablesefficient genomic diversification in mammalian cells,” the latest research results, reported on the use of Targeted cytosine deaminase to achieve high efficiency and high flux in the body of new method for DNA base editting.
Single nucleotide diversity is the main source of genetic diversity, the power of molecular evolution and the direct cause of many diseases. However, due to the height of the mammalian genome stability, it is difficult to efficiently in mammalian cells and high flux induction single nucleotide mutation, and then studies the function of these mutations. Although through the CRISPR editing techniques such as gene, can realize more efficient cut DNA and gene knockout, but due to the inefficient homologous recombination (HDR), existing CRISPR techniques for body building is still in the stage of inefficient single nucleotide mutations.
Targeting AID mediated nucleotides (TAM) this kind of new research methods, could change all that. Different from most of the somatic cell genome, adaptive immune system in the process of lymphocyte development can efficiently edit, to efficient mutation of antigen receptors. Libraries have almost unlimited antigen receptors, with protection against possible pathogen invasion. Inspired by the mechanism of mutation, a doctoral student Zhang Jiayuan under the guidance of Chang Xing researcher found that when the nuclease defect Cas9 protein and high frequency mutation induced antibodies cytosine deaminase AID after the fusion, in sgRNA targeted genome DNA, cytosine and guanine can randomly to the other three bases. This new method can be within the cells on specific DNA sequences to diversify, complete genetic screening, so as to analyze the function of single nucleotide mutation. At the same time in a kind of peptide inhibitors, dCas9 - AID can induce specific cytosine to thymine, realization of single-base precise editing. Further evidence, the team take advantage of this method can effectively simulate the heterogeneity of drug resistance in tumor cells in the body, to predict possible mutations in tumor resistance, research and improvement of small molecule inhibitors and small molecule and the interaction of protein targets. The results of the study of molecular evolution, gene therapy and gene regulation in single base level analysis components in areas such as providing a new method.
The work got department youth one thousand plan, the ministry of science and technology, national natural science foundation, and science and technology commission of Shanghai.
DCas9 - AID fusion protein was sgRNA recruit to the corresponding genome DNA, random point mutation induction cytosine and guanine, so as to realize the diversification of specific DNA sequences in the body, through genetic screening, high throughput analysis of single nucleotide mutation function.