CRISPR gRNA library screen of the HIV-1 genome

 描述(由申请人提供)：艾滋病毒-1感染仍然是一个大流行问题。治愈的目标仍然难以实现，需要新的治疗方法。最近提出的一项技术利用了CRISPR细菌免疫系统，其中Cas9蛋白被引导RNA(GRNA)靶向并诱导序列特异性双链断裂。这种DNA损伤通常通过容易出错的非同源末端连接途径修复，该途径通常在修复连接处引入插入和缺失。这项技术已经被提出用来删除HIV-1前病毒。然而，CRISPR靶向HIV-1基因组的全部影响尚不清楚。只对gRNA的一小部分进行了减少HIV-1复制的测试。病毒产生耐药突变所需的时间尚未揭示，整个HIV-1基因组中gRNAs的相对效率也没有得到系统的研究。该提案包含两个高度聚焦的具体目标：1.开发全基因组HIV-1 CRISPR/Cas9 gRNA文库，用于HIV-1筛查和2.绘制HIV-1全基因组突变图谱以响应CRISPR/Cas9选择压力。我们将生成一个针对整个HIV-1 B亚型基因组的CRISPR gRNA文库，不包括env基因的可变区。这个文库的特点是相对减少了HIV-1的复制和产生抗药性的时间。耐药株将通过NextGen测序进行分析，以揭示突变的性质和HIV-1突变的热图，以响应在整个病毒基因组上均匀施加的选择压力。最终，这笔探索性赠款将为探索HIV-1在选择性压力下的进化提供一个量化和适应性强的平台。这些数据将为CRISPR作为一种针对HIV-1前病毒的新型治疗方法的使用提供重要的见解。

 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.