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In situ functional genomics to understand transcriptional regulation

原位功能基因组学了解转录调控

基本信息

批准号：
10199425
负责人：
Neville Sanjana
金额：
$ 51.86万
依托单位：
NEW YORK GENOME CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-01 至 2021-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10199425
关键词：
2019-nCoV Bar Codes Binding Biochemical Biological Biological Assay Biological Models Blood Circulation COVID-19 COVID-19 pandemic CRISPR screen Capsid Proteins Cells China Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Colon Complement DNA DNA sequencing Databases Development Digestion Disease Outbreaks Epitopes Genetic Screening Genome Human Hydrophobicity Immunoblotting In Situ In Vitro Infection Interferometry Kinetics Lead Length Libraries Liver Lung Measures Membrane Mutation National Institute of Allergy and Infectious Disease Nucleic Acids Pathway interactions Peptide Hydrolases Peptides Point Mutation Population Prevalence Proteins Proteolytic Processing Reporting Research Resistance Resources Retroviridae Role SARS coronavirus Severe Acute Respiratory Syndrome Site Subfamily lentivirinae Testing Tissues Transcriptional Regulation Vaccines Variant Viral Viral Cytopathogenic Effect Viral Genome Viral Load result Viral Vector Virion Virulence Virus Visualization base cell type cellular transduction fitness functional genomics gain of function in vivo innovation novel nuclease premature protein function receptor receptor binding tool transduction efficiency transmission process viral transmission

项目摘要

PROJECT SUMMARY The COVID-19 pandemic has taken the lives of nearly 500,000 people worldwide in the span of a few months. Recently, a novel isolate of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has emerged and rapidly surpassed others in prevalence, including the original SARS-CoV-2 isolate from Wuhan, China. This Spike variant is a defining feature of the most prevalent clade (A2a) of SARS-CoV-2 genomes worldwide and, recently, we and others have demonstrated this variant leads to virions with an ~8-fold increase in human cell transduction. This is the first experimental evidence of a SARS-CoV-2 population variant acting in a gain-of-function manner. Although there are hundreds of Spike variants now in circulation, we lack tools for high-throughput characterization of these variants and their virulence. Here, we propose to develop a massively-parallel, high- throughput approach to test all Spike variants using a pooled forward genetic screen, examine the impact of these mutations on proteolytic cleavage of Spike and on ACE2 receptor binding kinetics, and validate changes in viral transduction with live SARS-CoV-2 via an innovative trans-complementation assay. Our proposed studies aim to understand the interactions between Spike protein variants and host (human) cell infection and their underlying biochemical mechanisms. This research will enable us to predict whether particular Spike variants can drive more serious COVID-19 outbreaks.

项目摘要 COVID-19疫情在短短数月内夺走了全球近50万人的生命。最近，一种携带刺突蛋白点突变（D 614 G）的SARS-CoV-2病毒新分离株，出现并迅速超过其他病毒，包括来自武汉的SARS-CoV-2原始分离株，中国这种刺突变异是SARS-CoV-2基因组中最普遍的进化枝（A2 a）的一个定义特征最近，我们和其他人已经证明这种变异导致病毒体增加约8倍在人类细胞转导中。这是SARS-CoV-2群体变异体作用于功能增益方式。虽然现在有数百种Spike变体在流通，但我们缺乏高通量的工具。这些变异体的特征及其毒力。在这里，我们建议发展一个平行的，高- 通量方法，使用合并正向遗传筛选检测所有刺突变体，检查这些突变对刺突蛋白水解切割和ACE 2受体结合动力学的影响，并验证了这些变化通过一种创新的反式互补分析，用活SARS-CoV-2进行病毒转导。我们提出的研究旨在了解刺突蛋白变体与宿主（人）之间的相互作用。细胞感染及其潜在的生化机制。这项研究将使我们能够预测特定的Spike变体可能导致更严重的COVID-19爆发。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Cancer genes disfavoring T cell immunity identified via integrated systems approach.

DOI：
10.1016/j.celrep.2022.111153
发表时间：
2022-08-02
期刊：
CELL REPORTS
影响因子：
8.8
作者：
Kishton, Rigel J.;Patel, Shashank J.;Decker, Amy E.;Vodnala, Suman K.;Cam, Maggie;Yamamoto, Tori N.;Patel, Yogin;Sukumar, Madhusudhanan;Yu, Zhiya;Ji, Michelle;Henning, Amanda N.;Gurusamy, Devikala;Palmer, Douglas C.;Stefanescu, Roxana A.;Girvin, Andrew T.;Lo, Winifred;Pasetto, Anna;Malekzadeh, Parisa;Deniger, Drew C.;Wood, Kris C.;Sanjana, Neville E.;Restifo, Nicholas P.
通讯作者：
Restifo, Nicholas P.

Profiling the genetic determinants of chromatin accessibility with scalable single-cell CRISPR screens.

DOI：
10.1038/s41587-021-00902-x
发表时间：
2021-10
期刊：
Nature biotechnology
影响因子：
46.9
作者：
Liscovitch-Brauer N;Montalbano A;Deng J;Méndez-Mancilla A;Wessels HH;Moss NG;Kung CY;Sookdeo A;Guo X;Geller E;Jaini S;Smibert P;Sanjana NE
通讯作者：
Sanjana NE

High-Throughput Screens of PAM-Flexible Cas9 Variants for Gene Knockout and Transcriptional Modulation.