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科学家开发出更高活性的腺嘌呤碱基编辑器
作者:小柯机器人 发布时间:2020/4/16 11:55:30

美国 Beam Therapeutics公司Giuseppe Ciaramella、Nicole M. Gaudelli等研究人员,合作开发了具有更高活性和更广应用前景的腺嘌呤碱基编辑器。相关论文于2020年4月13日在线发表于《自然—生物技术》。

研究人员使用腺苷脱氨酶变体库进一步进化了ABE7.10,从而产生了ABE8。与ABE7.10相比,在NGG原型间隔子相邻基序(PAM)位置,ABE8在原型间隔位A5-A7处的编辑度高约1.5倍,在位置A3-A4和A8-A10处的编辑度约高3.2倍。非NGG PAM变体的总体目标编辑效率比ABE7.10高约4.2倍。
 
在人类CD34+细胞中,ABE8可以在γ-珠蛋白基因HBG1和HBG2的启动子上重建天然等位基因,效率高达60%,从而使得胎儿血红蛋白持续存在。在原代人类T细胞中,ABE8可实现98-99%的靶标修饰,当在三个基因座上多重修饰时,该高效率仍得以维持。
 
作为信使RNA传递,ABE8在基因组DNA中不会诱导显著水平的不依赖单个向导RNA(sgRNA)的脱靶腺嘌呤脱氨,而在细胞mRNA中只产生非常低水平的腺嘌呤脱氨。
 
据了解,基础的腺嘌呤碱基编辑器(例如,ABE7.10)能够编辑A•T到G•C的点突变,但在修饰原代人类细胞中的基因座时,编辑效率可能很低。
 
附:英文原文

Title: Directed evolution of adenine base editors with increased activity and therapeutic application

Author: Nicole M. Gaudelli, Dieter K. Lam, Holly A. Rees, Noris M. Sol-Esteves, Luis A. Barrera, David A. Born, Aaron Edwards, Jason M. Gehrke, Seung-Joo Lee, Alexander J. Liquori, Ryan Murray, Michael S. Packer, Conrad Rinaldi, Ian M. Slaymaker, Jonathan Yen, Lauren E. Young, Giuseppe Ciaramella

Issue&Volume: 2020-04-13

Abstract: The foundational adenine base editors (for example, ABE7.10) enable programmable AT to GC point mutations but editing efficiencies can be low at challenging loci in primary human cells. Here we further evolve ABE7.10 using a library of adenosine deaminase variants to create ABE8s. At NGG protospacer adjacent motif (PAM) sites, ABE8s result in ~1.5× higher editing at protospacer positions A5–A7 and ~3.2× higher editing at positions A3–A4 and A8–A10 compared with ABE7.10. Non-NGG PAM variants have a ~4.2-fold overall higher on-target editing efficiency than ABE7.10. In human CD34+ cells, ABE8 can recreate a natural allele at the promoter of the γ-globin genes HBG1 and HBG2 with up to 60% efficiency, causing persistence of fetal hemoglobin. In primary human T cells, ABE8s achieve 98–99% target modification, which is maintained when multiplexed across three loci. Delivered as messenger RNA, ABE8s induce no significant levels of single guide RNA (sgRNA)-independent off-target adenine deamination in genomic DNA and very low levels of adenine deamination in cellular mRNA.

DOI: 10.1038/s41587-020-0491-6

Source: https://www.nature.com/articles/s41587-020-0491-6

期刊信息

Nature Biotechnology:《自然—生物技术》,创刊于1996年。隶属于施普林格·自然出版集团,最新IF:31.864
官方网址:https://www.nature.com/nbt/
投稿链接:https://mts-nbt.nature.com/cgi-bin/main.plex