A CRISPR-Cas9 screen for novel proteins required for induction of CYP1A1 by AHR

 说明(申请人提供)：环境污染物2，3，7，8-四氯二苯并对二恶英(TCDD)具有包括致癌在内的大量毒性作用。TCDD和相关多氯化合物的所有毒性效应都是由芳烃受体(AHR)介导的，并依赖于AHR对尚未完全识别的下游基因的转录激活。AHR还通过多环芳烃(如苯并[a]芘)介导致癌作用，这些化合物是烟草烟雾和烟雾中的重要致癌物质。细胞色素P1A1、细胞色素P1A2和细胞色素P1B1在许多组织中被大量诱导，并可能通过将它们代谢成亲电的衍生物来介导多环芳烃的致癌作用。在结合激动剂后，AHR移位到细胞核，并与芳烃核转位蛋白(ArnT)形成二聚体，然后与反应基因的增强子区域结合，从而导致其转录激活。在目前的提案中，我们将寻求识别在以下方面发挥作用的新基因产品 通过筛选CRISPR-Cas9文库，研究AHR对基因转录的诱导。CRISPR-Cas9系统可以通过在引导RNAs(GRNAs)内合成20个核苷酸序列在特定的基因组座位上诱导DNA双链断裂，当针对基因的编码区时，这些序列可以产生缺失或插入，导致移码突变，导致二倍体细胞中两个等位基因的功能丧失。有两个具体目标。在具体目标1中， 将通过使用GeCKOv2文库来鉴定诱导CYP1A1所需的基因，该文库针对20,611个小鼠基因(因此几乎所有蛋白质编码基因)和1,178个microRNAs。该文库包含在单个高滴度慢病毒载体(LentiCRISPRv2)中，该载体 将其转导入小鼠肝癌细胞系HEPA-1。转导细胞将在苯并[a]芘中选择10天。(苯并[a]芘抗性克隆失去了依赖AHR的细胞色素P1A1的诱导)。然后对整合的gRNA进行聚合酶链式反应扩增和测序。那些由一个以上的gRNA代表的基因，和/或在一个以上的转导培养中鉴定的基因，和/或高频代表的基因，很可能代表真正的阳性。《特定目标2》将验证来自屏幕的点击。每个目的基因的两个gRNAs将被插入到lentiCRISPRv2中，以确定它们是否赋予苯并[a]芘抗性，减少TCDD对CYP1A1的诱导，并在转导到HEPA-1细胞时降低AHR和Arnt的表达。相应的内源基因将在某些转导子中进行测序，以查看它们是否包含缺失或插入。确认命中的特性将在未来的R01应用中继续进行。这些HITS可能包括影响AHR或ARNT表达或功能的基因产物，和/或转录激活CYP1A1所需的基因产物。因此，目前的建议为全面分析AHR依赖的基因转录诱导机制奠定了基础，并可能导致减少AHR配体的有害影响的策略。

 DESCRIPTION (provided by applicant): The environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a large number of toxic effects, including carcinogenicity. All the toxic effects of TCDD and of related polychlorinated compounds are mediated by the Aryl Hydrocarbon Receptor (AHR) and depend upon transcriptional activation by the AHR of not yet fully identified downstream genes. AHR also mediates carcinogenesis by polycyclic aromatic hydrocarbons, (e.g. benzo[a]pyrene) which are important carcinogens in tobacco smoke and smog. CYP1A1, CYP1A2 and CYP1B1 are massively induced in many tissues and are likely responsible for mediating the carcinogenic effects of PAHs, via metabolizing them to electrophilic derivatives. After binding agonist, the AHR translocates to the nucleus and forms a dimer with the aryl hydrocarbon nuclear translocator (ARNT) protein, which then binds to the enhancer regions of responsive genes, thereby leading to their transcriptional activation. In the current proposal we will seek to identify novel gene products that play roles in the induction of gene transcription by AHR, by screening a CRISPR-Cas9 library. The CRISPR-Cas9 system can induce DNA double strand breaks at specific genomic loci through synthetic 20 nucleotide sequences within guide RNAs (gRNAs), which when targeted to coding regions of genes can generation of deletions or insertions, resulting in frameshift mutations which lead to loss of function at both alleles in a diploid cell. There are two specific aims. In Specific Aim 1, genes required for CYP1A1 induction will be identified by using the GeCKOv2 library, which targets 20,611 mouse genes (and thus nearly all protein coding genes) and also 1,178 microRNAs. The library is contained in a single high titer lentiviral vector (lentiCRISPRv2), which will be transduced into the mouse hepatoma cell line, Hepa-1. The transduced cells will be selected for 10 days in benzo[a]pyrene. (Benzo[a]pyrene-resistant clones exhibit loss of AHR-dependent induction of CYP1A1). PCR amplification of the integrated gRNAs and their sequencing will then be performed. Those genes represented by more than one gRNA, and/or identified in more than one transduced culture, and/or represented at high frequency, are likely to represent true positives. Specific Aim 2 will validate hits from the screen. Two gRNAs for each gene of interest will be inserted into lentiCRISPRv2 to determine if they confer benzo[a]pyrene resistance, reduce TCDD induction of CYP1A1, and reduce AHR and ARNT expression upon transduction into Hepa-1 cells. The corresponding endogenous genes will be sequenced in certain transductants to see if they contain deletions or insertions. Characterization of confirmed hits will be pursued in a future R01 application. Such hits may include gene products that affect AHR or ARNT expression or function, and/or that are required for transcriptional activation of CYP1A1. The current proposal therefore lays the foundations for a comprehensive analysis of the mechanism of AHR-dependent induction of gene transcription, and may lead to strategies for reducing the harmful effects of ligands of the AHR.