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

 描述（申请人提供）：环境污染物2，3，7，8-四氯二苯并对二恶英（TCDD）具有大量毒性作用，包括致癌性。四氯二苯并对二恶英和相关多氯化合物的所有毒性作用都是由芳烃受体（AHR）介导的，并取决于尚未完全确定的下游基因的AHR的转录激活。AHR还通过多环芳烃（例如苯并[a]芘）介导致癌作用，多环芳烃是烟草烟雾和烟雾中的重要致癌物。CYP 1A 1、CYP 1A 2和CYP 1B 1在许多组织中被大量诱导，并且可能负责介导多环芳烃的致癌作用，通过将它们代谢为亲电子衍生物。在结合激动剂后，AHR易位到细胞核并与芳烃核易位蛋白（ARNT）形成二聚体，然后与应答基因的增强子区域结合，从而导致其转录激活。在目前的建议中，我们将寻求确定新的基因产物，这些基因产物在以下方面发挥作用： AHR通过筛选CRISPR-Cas9文库诱导基因转录。CRISPR-Cas9系统可以通过向导RNA（gRNA）内的合成的20个核苷酸序列在特定基因组基因座处诱导DNA双链断裂，当靶向基因的编码区时，其可以产生缺失或插入，导致移码突变，其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。具体目标1、 CYP 1A 1诱导所需的基因将通过使用GeCKOv 2文库来鉴定，该文库靶向20，611个小鼠基因（因此几乎所有蛋白质编码基因）和1，178个microRNA。该文库包含在单个高滴度慢病毒载体（lentiCRISPRv 2）中， 将被转导到小鼠肝癌细胞系Hepa-1中。将在苯并[a]芘中选择转导的细胞10天。（苯并[a]芘抗性克隆表现出CYP 1A 1的AHR依赖性诱导丧失）。然后将对整合的gRNA进行PCR扩增并对其进行测序。由多于一种gRNA代表的和/或在多于一种转导培养物中鉴定的和/或以高频率代表的那些基因可能代表真阳性。特定目标2将验证屏幕上的命中。将每个感兴趣基因的两个gRNA插入到lentiCRISPRv 2中，以确定它们是否赋予苯并[a]芘抗性，减少CYP 1A 1的TCDD诱导，以及在转导到Hepa-1细胞中时减少AHR和ARNT表达。将在某些转导子中对相应的内源基因进行测序，以查看它们是否含有缺失或插入。将在未来的R 01申请中对确认的命中进行表征。这些命中可能包括影响AHR或ARNT表达或功能和/或CYP 1A 1转录激活所需的基因产物。因此，目前的建议奠定了基础，AHR依赖的诱导基因转录的机制进行全面分析，并可能导致战略，减少有害影响的配体的AHR。

 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.