CRISPR gRNA library screen of the HIV-1 genome

HIV-1 基因组的 CRISPR gRNA 文库筛选

• 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
• 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

 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，需要新的治疗方法。最近提出的技术之一利用CRISPR细菌免疫系统，其中蛋白质Cas9被引导RNA（gRNA）靶向并诱导序列特异性双链断裂。这种DNA损伤通常通过易错非同源末端连接途径修复，其通常在修复连接处引入插入和缺失。该技术已被提出用于删除HIV-1前病毒。然而，CRISPR靶向HIV-1基因组的全部效果尚不清楚。只有一小部分gRNA被测试用于减少HIV-1复制。病毒产生耐药性突变所需的时间尚未被揭示，整个HIV-1基因组中gRNA的相对效率尚未被系统地探索。该提案包含两个高度集中的具体目标：1。开发用于HIV-1筛选的全基因组HIV-1 CRISPR/Cas9 gRNA文库，以及2.）绘制HIV-1全基因组突变谱以响应CRISPR/Cas9选择压力。我们将生成一个CRISPR gRNA文库，靶向整个HIV-1亚型B基因组，不包括env基因的可变区。该文库将表征HIV-1复制的相对减少和产生耐药性的时间。耐药菌株将通过NextGen测序进行分析，以揭示突变的性质和HIV-1突变性的热图，以响应在病毒基因组中均匀施加的选择压力。最终，这项探索性资助将为探索HIV-1在选择压力下的进化提供一个定量和适应性强的平台。这些数据将为CRISPR作为靶向HIV-1前病毒的新型治疗方法的使用提供重要见解。