香港大学教授黄兆麟（Alan S. L. Wong）博士课题组通过其命名的CombiSEAL技术筛选出一个SpCas9突变体，该突变体在不损失活性及靶标位点选择范围的前提下对基因组编辑的特异性更强。 该项研究成果于2019年8月2日发表于《自然—方法学》上。

该课题组研究人员建立了一个高通量的平台（称之为CombiSEAL），通过组合修饰实现被条码标记的蛋白变异的可扩展组装和并行表征。通过系统地描述一个被广泛使用的链球菌Cas9（SpCas9）核酸酶包含 948个组合的突变体库，优化其在人类细胞基因组编辑活动。评估SpCas9编辑能力是通过检测多个靶标（on-target）和非靶标（off-target）位点，从而加快了优化变体的识别速度并有助于对突变上位的研究。最终，成功筛选出不损失活性和广泛的定位范围并具有增强的编辑特异性的Opti-SpCas9。该平台广泛适用于通过组合修饰的工程蛋白。

据介绍，多个突变对蛋白质功能的综合效应是很难预测的，因此，对大量蛋白质序列变异进行功能性评估的能力将在蛋白质工程中具有实际意义。

附：英文原文

Title: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9

Author: Gigi C. G. Choi, Peng Zhou, Chaya T. L. Yuen, Becky K. C. Chan, Feng Xu, Siyu Bao, Hoi Yee Chu, Dawn Thean, Kaeling Tan, Koon Ho Wong, Zongli Zheng, Alan S. L. Wong

Issue&Volume: Volume 16 Issue 8

Abstract: The combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.

DOI: 10.1038/s41592-019-0473-0

Source:https://www.nature.com/articles/s41592-019-0473-0