Saturation profiling of drug-resistant genetic variants using prime editing

IF 33.1 1区 生物学 Q1 BIOTECHNOLOGY & APPLIED MICROBIOLOGY Nature biotechnology Pub Date : 2024-11-12 DOI:10.1038/s41587-024-02465-z
Younggwang Kim, Hyeong-Cheol Oh, Seungho Lee, Hyongbum Henry Kim
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Abstract

Methods to characterize the functional effects of genetic variants of uncertain significance (VUSs) have been limited by incomplete coverage of the mutational space. In clinical oncology, drug resistance arising from VUSs can prevent optimal treatment. Here we introduce PEER-seq, a high-throughput method based on prime editing that can evaluate the functional effects of single-nucleotide variants (SNVs). PEER-seq introduces both intended SNVs and synonymous marker mutations using prime editing and deep sequences the endogenous target regions to identify the introduced SNVs. We generate and functionally evaluate 2,476 SNVs in the epidermal growth factor receptor gene (EGFR), including 99% of all possible variants in the canonical tyrosine kinase domain. We determined resistance profiles of 95% of all possible EGFR protein variants encoded in the whole tyrosine kinase domain against the common tyrosine kinase inhibitors afatinib, osimertinib and osimertinib in the presence of the co-occurring substitution T790M, in PC-9 cells. Our study has the potential to substantially improve the precision of therapeutic choices in clinical settings.

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利用素材编辑技术对耐药性基因变异进行饱和分析
由于变异空间覆盖不全,描述意义不确定遗传变异(VUS)功能影响的方法一直受到限制。在临床肿瘤学中,VUSs 引起的耐药性会阻碍最佳治疗。在这里,我们介绍一种基于素材编辑的高通量方法 PEER-seq,它可以评估单核苷酸变异(SNV)的功能效应。PEER-seq 利用质粒编辑引入预期 SNV 和同义标记突变,并对内源性靶区进行深度测序,以识别引入的 SNV。我们在表皮生长因子受体基因(EGFR)中产生了 2,476 个 SNV,并对其进行了功能评估,其中包括典型酪氨酸激酶结构域中所有可能变异的 99%。我们确定了整个酪氨酸激酶域编码的所有可能的表皮生长因子受体蛋白变体中的 95% 在 PC-9 细胞中对常见酪氨酸激酶抑制剂阿法替尼、奥西莫替尼和奥西莫替尼(存在共存替代物 T790M)的耐药性特征。我们的研究有望大幅提高临床治疗选择的精确性。
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来源期刊
Nature biotechnology
Nature biotechnology 工程技术-生物工程与应用微生物
CiteScore
63.00
自引率
1.70%
发文量
382
审稿时长
3 months
期刊介绍: Nature Biotechnology is a monthly journal that focuses on the science and business of biotechnology. It covers a wide range of topics including technology/methodology advancements in the biological, biomedical, agricultural, and environmental sciences. The journal also explores the commercial, political, ethical, legal, and societal aspects of this research. The journal serves researchers by providing peer-reviewed research papers in the field of biotechnology. It also serves the business community by delivering news about research developments. This approach ensures that both the scientific and business communities are well-informed and able to stay up-to-date on the latest advancements and opportunities in the field. Some key areas of interest in which the journal actively seeks research papers include molecular engineering of nucleic acids and proteins, molecular therapy, large-scale biology, computational biology, regenerative medicine, imaging technology, analytical biotechnology, applied immunology, food and agricultural biotechnology, and environmental biotechnology. In summary, Nature Biotechnology is a comprehensive journal that covers both the scientific and business aspects of biotechnology. It strives to provide researchers with valuable research papers and news while also delivering important scientific advancements to the business community.
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