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科学家研发出可用于人单碱基突变筛选的碱基编辑器
作者:小柯机器人 发布时间:2021/2/20 10:35:04

美国麻省理工博德研究所John G. Doench小组近日取得一项新成果。他们使用碱基编辑器筛选实现了对人类突变体的大规模并行评估。相关论文于2021年2月18日发表在国际学术期刊《细胞》上。

研究人员在大规模筛选中利用CRISPR-Cas9胞嘧啶碱基编辑器有效地分析了哺乳动物细胞内源性基因座的变异体。研究人员在正向和负向选择筛选中对碱基编辑器的性能进行基准测试,并以高精度识别出BRCA1和BRCA2中已知的功能丧失突变。

为了证明该碱基编辑器筛选可用于探测小分子与蛋白质的相互作用,研究人员对BH3模拟物和PARP抑制剂进行了筛选,发现了药物敏感性或耐药性的点突变。研究还创建了一个单向导RNA(sgRNA)库,该库预计可在3,584个基因中生成52,034个ClinVar变体,并在存在细胞应激因子的情况下进行筛选,从而鉴定了许多DNA损伤修复基因中的功能丧失变体。研究人员估计该筛选方法可广泛用于遗传变异筛选,并具有便捷和可扩展性的特点。

研究人员表示,了解单核苷酸突变的影响对于揭示疾病的遗传基础至关重要,但是表征变异的技术却是有限的。

附:英文原文

Title: Massively parallel assessment of human variants with base editor screens

Author: Ruth E. Hanna, Mudra Hegde, Christian R. Fagre, Peter C. DeWeirdt, Annabel K. Sangree, Zsofia Szegletes, Audrey Griffith, Marissa N. Feeley, Kendall R. Sanson, Yossef Baidi, Luke W. Koblan, David R. Liu, James T. Neal, John G. Doench

Issue&Volume: 2021/02/18

Abstract: Understanding the functional consequences of single-nucleotide variants is criticalto uncovering the genetic underpinnings of diseases, but technologies to characterizevariants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooledscreens to scalably assay variants at endogenous loci in mammalian cells. We benchmarkthe performance of base editors in positive and negative selection screens, identifyingknown loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe smallmolecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors,identifying point mutations that confer drug sensitivity or resistance. We also createa library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variantsin 3,584 genes and conduct screens in the presence of cellular stressors, identifyingloss-of-function variants in numerous DNA damage repair genes. We anticipate thatthis screening approach will be broadly useful to readily and scalably functionalizegenetic variants.

DOI: 10.1016/j.cell.2021.01.012

Source: https://www.cell.com/cell/fulltext/S0092-8674(21)00012-X

期刊信息
Cell:《细胞》,创刊于1974年。隶属于细胞出版社,最新IF:36.216
官方网址:https://www.cell.com/