Research and development of new dual base editing tools to aid gene therapy

Following the report in May this year of hyperactive cytosine base editor hyCBEmax, the Li Wei Group and Liu Mingyao Of the School of Life Sciences of East China Normal University published a paper online in Nature-Biotech on the evening of June 1st, showing a new tool for gene editing, A-C-BEmax. The tool breaks down the limitations of existing base editor base, innovatively develops a new two-function base editor, and enables the conversion of two bases, cytosine and adenine, providing new tools for basic research and treatment of genetic disorders such as β-thalassemia.About 58 percent of genetic diseases in humans are caused by base mutations, according to ClinVar. However, the existing base editor can only catalyce the conversion of a single type of base, it is difficult to treat genetic diseases caused by two base mutations, and a single base product also restricts its application in basic research such as molecular evolution and saturation mutation screening. Therefore, the development of an efficient base editor that can catalyt both types of base conversion is not only a conceptual innovation, but also a great enrichment of base editing tools.
Li and Liu Mingyao combined cytosine deaminase (hAID) and adenine deaminase (ecTadA-ecTadA) with enzyme-active defective Cas9 to build a carrier of 5 fusion methods.
through mammalian cell testing, screening to the optimal fusion method, while achieving adenine (A) to ostrich (G) and cytosine (C) to thymus (T) base conversion. The breakthrough cheered the team, but the low conversion efficiency also left researchers feeling there was room for improvement.
In order to further improve editing efficiency, the team obtained an efficient dual-base editor, named A-C-BEmax, through multiple rounds of optimization such as cryptonogens, nuclear positioning signals, linker sequences, and UGI, a fusion of urine-based glycosylase inhibitors. At the same time, the team comprehensively evaluated the performance of this new tool. Through testing at 28 targets in the human cell line, it was found that the editing window for cytosine in A-C-BEmax was tripled, the editing activity was increased by 1.9-14 times, the off-target effect was lower, and the efficiency of simultaneous A/C mutations was up to 30%.
the unique properties of the A-C-BEmax, which also makes it unique in gene therapy applications. β is a high-risk genetic disease in the south of the country and in the tropical regions of the world. Studies have shown that improving fetal hemoglobin to replace damaged adult hemoglobin is promising to cure β hemoglobin disease, which includes β thalassemia and sickle-like anemia β The researchers delivered the A-C-BEmax and wizard RNA to red blood cell precursor cells (HUDEP-2). The results showed that the A-C-BEmax was able to effectively destroy the fetal hemoglobin gene HBG1/2 promoter region transcription inhibitor BCL11A binding site and at the same time create a new transcription activation factor GATA1 binding site, which caused the fetal hemoglobin to rise significantly above the theoretical cure level, which is of great significance for the treatment of β thalassemia and sickle cell anemia. This work demonstrates the great potential of the new dual-base editing system, A.C.-BEmax, for the precise treatment of genetic diseases.competition in the field of gene editing research. On June 1st alone, Nature Biotechnology reported similar research results in the field of gene editing tools developed by three global research groups, and similar studies by Gao Caixia and Li Jiayang of the Chinese Academy of Sciences, published online earlier this year in the same journal, a total of four similar gene editing tools were reported, albeit with different names but similar functions.
From the data published in the paper, the editing efficiency, product purity, saturation of base mutations and mutation types have been significantly improved, and no DNA off-targeting has been found, and RNA levels have been significantly reduced, which is a very efficient dual-functional base editing tool.
", A/C simultaneously mutated and differentiated HUDEP-2 cells had higher HBG expression levels than cells that produced C-to-T base editing alone. Zhang Xiaohui, co-lead author of the paper and a doctoral student at the School of Life Sciences at East China University, explains that this means that the new dual-base editing system, A-C-BEmax, has great potential for the precise treatment of β-hemoglobin disease.
"s single-clone cell experiments, which screened different genotypes, also demonstrated that A.C.-BEmax can analyze the effects of DNA sequences on gene transcription in the dimensions of a single base. Zhu Biyun, co-first author of the paper and a doctoral student at the School of Life Sciences at East China University, added.
experts say the new dual-base editing tool is significant for gene therapy, species improvement and molecular evolution, disease modeling, and drug resistance screening. (Source: Huang Xinbuye, China Science Journal)
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