CRISPR gRNA library screen of the HIV-1 genome

HIV-1 基因组的 CRISPR gRNA 文库筛选

基本信息

批准号：
9064991
负责人：
KRISTINE E YODER
金额：
$ 19.25万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-08-15 至 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9064991
关键词：
Anti-Retroviral Agents Antiretroviral drug resistance CCR5 gene CD4 Positive T Lymphocytes CRISPR library CRISPR/Cas technology Cell Line Clinic Clustered Regularly Interspaced Short Palindromic Repeats Cultured Cells DNA Damage Data Databases Development Evolution Fostering Frequencies Genetic Recombination Genome Goals Grant Guide RNA HIV-1 Heating Human Immune system Immunity Immunoglobulin Variable Region Infection Libraries Location Maps Methods Mutation Nature Neisseria meningitidis Nonhomologous DNA End Joining Orthologous Gene Pathway interactions Pharmaceutical Preparations Production Proteins Proviruses RNA library RNA-Directed DNA Polymerase Receptor Gene Resistance Resistance development Retroviridae Site Streptococcus pyogenes System Technology Testing Therapeutic Therapeutic Intervention Time Translating Viral Viral Genome Virus Virus Replication base cell type deep sequencing endonuclease env Genes genome editing improved in vivo insertion/deletion mutation insight new technology new therapeutic target next generation sequencing novel novel therapeutic intervention pandemic disease pressure public health relevance repaired resistance mutation resistant strain response screening targeted treatment tool whole genome

项目摘要

DESCRIPTION (provided by applicant): HIV-1 infection continues to be a pandemic problem. The goal for a cure remains elusive and will require novel therapeutic approaches. One of the recently proposed technologies utilizes the CRISPR bacterial immunity system where the protein Cas9 is targeted by a guide RNA (gRNA) and induces a sequence specific double strand break. Such DNA damage is generally repaired by the error prone non homologous end joining pathway which typically introduces insertions and deletions at the repair junction. This technology has been proposed to delete the HIV-1 provirus. However, the full effects of CRISPR targeting of the HIV-1 genome are unknown. Only small subsets of gRNAs have been tested for reduction of HIV-1 replication. The time required for the virus to generate resistance mutations has not been revealed and the relative efficiency of gRNAs throughout the HIV-1 genome has not been systematically explored. This proposal contains two highly focused Specific Aims: 1.) Develop a whole genome HIV-1 CRISPR/Cas9 gRNA library for use in an HIV-1 screen and 2.) Map the HIV-1 whole genome mutation profile in response to CRISPR/Cas9 selective pressure. We will generate a library of CRISPR gRNAs targeting the entire HIV-1 subtype B genome, excluding the variable regions of the env gene. This library will be characterized for both the relative reduction in HIV-1 replication and the time to develop resistance. Resistant strains will be analyzed by NextGen sequencing to reveal the nature of the mutations and a heat map of HIV-1 mutability in response to selective pressure applied evenly across the viral genome. Ultimately, this exploratory grant will provide a quantitative and adaptable platform for probing the evolution of HIV-1 in response to selective pressure. These data will yield significant insight into the use of CRISPR as a novel therapeutic targeting the HIV-1 provirus.

描述（由申请人提供）：HIV-1 感染仍然是一个大流行问题，治愈的目标仍然难以捉摸，并且需要新的治疗方法，其中一项最近提出的技术利用了针对蛋白质 Cas9 的 CRISPR 细菌免疫系统。这种 DNA 损伤通常通过容易出错的非同源末端连接途径进行修复，该途径通常在修复连接处引入插入和缺失。然而，CRISPR 靶向 HIV-1 基因组的全部效果尚不清楚，仅测试了一小部分 gRNA 减少 HIV-1 复制所需的时间。突变尚未被揭示，并且 gRNA 在整个 HIV-1 基因组中的相对效率尚未得到系统探索。该提案包含两个高度集中的具体目标： 1.) 开发一个全基因组 HIV-1 CRISPR/Cas9 gRNA 文库。一个HIV-1 筛选和 2.) 绘制针对 CRISPR/Cas9 选择压力的 HIV-1 全基因组突变图谱我们将生成针对整个 HIV-1 B 亚型基因组（不包括可变区）的 CRISPR gRNA 文库。该文库将通过 NextGen 测序来分析 HIV-1 复制的相对减少和产生耐药性的时间，以揭示突变的性质和 HIV-1 突变的热图。对选择压力的反应最终，这项探索性资助将提供一个定量且适应性强的平台，用于探索 HIV-1 响应选择性压力的进化。 HIV-1原病毒。