美国哈佛大学David R. Liu课题组发现了具有改进的靶序列相容性和更高活性的编辑器。 该项研究成果发表在2019年9月出版的《自然—生物技术》上。

研究人员利用噬菌体辅助连续进化（PACE）来生成具有改进的靶序列相容性和更高活性的编辑器。研究人员利用BE-PACE进化胞嘧啶碱基编辑器（CBE），克服了CBE靶序列的背景限制。改进的CBE，evoAPOBEC1-BE4max，在GC环境中编辑胞嘧啶的效率高达26倍，这是一个不利用野生型APOBEC1脱氨酶的的环境，同时在所有其他测试序列环境中保持有效编辑。另一种进化的脱氨酶evoFERNY比APOBEC1小29％，并且在所有测试序列环境中都可以有效编辑。研究人员还开发了基于CDA1脱氨酶的CBE，在难编辑的靶位点具有更高的编辑效率。最后，研究人员使用来自进化的CBE的数据来阐明脱氨酶活性、碱基编辑效率、编辑窗口宽度和副产物形成之间的关系。这些发现建立了一个快速发展的基础编辑系统，为进一步推进应用奠定了基础。

据了解，碱基编辑使用靶向催化受损的CRISPR蛋白的DNA修饰酶来精确地进行点突变。

附：英文原文

Title: Continuous evolution of base editors with expanded target compatibility and improved activity

Author: Benjamin W. Thuronyi, Luke W. Koblan, Jonathan M. Levy, Wei-Hsi Yeh, Christine Zheng, Gregory A. Newby, Christopher Wilson, Mantu Bhaumik, Olga Shubina-Oleinik, Jeffrey R. Holt, David R. Liu

Issue&Volume:Volume 37 Issue 9

Abstract: Base editors use DNA-modifying enzymes targeted with a catalytically impaired CRISPR protein to precisely install point mutations. Here, we develop phage-assisted continuous evolution of base editors (BEPACE) to improve their editing efficiency and target sequence compatibility. We used BEPACE to evolve cytosine base editors (CBEs) that overcome target sequence context constraints of canonical CBEs. One evolved CBE, evoAPOBEC1-BE4max, is up to 26-fold more efficient at editing cytosine in the GC context, a disfavored context for wild-type APOBEC1 deaminase, while maintaining efficient editing in all other sequence contexts tested. Another evolved deaminase, evoFERNY, is 29% smaller than APOBEC1 and edits efficiently in all tested sequence contexts. We also evolved a CBE based on CDA1 deaminase with much higher editing efficiency at difficult target sites. Finally, we used data from evolved CBEs to illuminate the relationship between deaminase activity, base editing efficiency, editing window width and byproduct formation. These findings establish a system for rapid evolution of base editors and inform their use and improvement.

DOI: 10.1038/s41587-019-0193-0

Source:https://www.nature.com/articles/s41587-019-0193-0