【作者】 李博；

【导师】 黄启来；

【作者基本信息】 山东大学 ， 细胞生物学， 2023， 博士

【摘要】 癌症已经成为人类在世界范围内的主要死亡原因,作为复杂性状疾病,挖掘、筛选以及注释癌症相关的遗传变异位点对于癌症的预测、诊断和治疗有着重要的意义。目前全基因组关联分析(GWAS)发现了大量的癌症风险变异位点。然而,GWAS发现的风险变异位点通常是变异频率大于1%的常见变异,不包括可能具有更大遗传效应的稀有变异位点。此外,GWAS发现的这些风险变异位点是否是导致疾病风险的因果位点还需要大量的功能性分析和筛选工作。大量研究表明,大部分风险变异位点主要通过改变特定转录因子的结合影响下游基因表达,进而导致疾病风险。因此,本项目从与癌症发生、发展有密切关系的转录因子出发,通过分析肿瘤临床样本ChIP-seq数据筛选影响转录因子结合的变异位点,并系统探索了通过影响关键转录因子结合的调控性变异位点。HOXB13转录因子在前列腺癌中发挥着重要调控功能,前期报道表明前列腺癌风险SNP位点rs339331通过介导HOXB13调控下游靶基因RFX6的表达,从而影响癌症风险。本研究从HOXB13转录因子的调控作用出发,全景式筛选介导HOXB13发挥调控作用的SNP位点。我们获取42个前列腺病人临床样本中的HOXB13 ChIP-seq数据,分析HOXB13染色质结合峰内存在的全部SNP位点,通过EEL算法以及ChIP-seq测序read富集程度的差异评估了不同等位基因与HOXB13的结合能力,筛选出能够影响HOXB13结合的变异位点。进一步,结合HOXB13敲低细胞的RNA-seq数据,根据临近基因或者相互作用基因的变化筛选出了 4814个潜在的由HOXB13介导的基因调控性SNP。随后通过大规模并行报告基因分析(MPRA)实验对4814个SNP的参考/变异等位基因共9628条DNA元件的调控活性进行评估,筛选出有调控活性而且具备等位基因差异的190个SNP位点。这些位点可能通过影响前列腺癌HOXB13基因调控重编程在疾病发生中发挥重要作用,此外,在这190个突变位点中有42.6%的SNV在人群中的变异频率低于1%,因此,此部分的研究工作发现的具有基因调控功能的变异位点能够与GWAS分析发现的风险位点互补,为全面发现与阐明癌症遗传易感风险的分子机制打下良好的基础。基因变异在人类疾病风险中发挥重要作用,解读基因变异的功能并阐明其致病机制具有重要意义。在研究过程中通常需要通过基因编辑构建基因变异模型,目前CRISPR/Cas9技术已经在各种生物中广泛用于靶向基因编辑。然而,如何准确检测基因编辑引入的基因变异仍然是有待解决的难题。本课题基于qPCR技术开发了一种快速、准确评估基因编辑的方法,称之为getPCR(genome editing test PCR)。该方法借助Taq DNA聚合酶在启动DNA复制过程中对引物3’末端核苷酸错配的敏感性,设计3’端跨Cas9切割位点3～5个碱基的可与未编辑野生型序列互补配对的indel敏感引物,用于区分发生基因编辑产生变异的序列,选择性扩增并定量野生型序列的比例,从而实现基因编辑的定量评估。该方法不仅可以准确检测NHEJ引入的随机indel突变,针对明确的编辑后目标序列设计引物,还可以准确定量HDR以及碱基编辑器方法引入的确定碱基变异,在基因编辑效率评估以及基因编辑单细胞克隆的基因分型分析中均具有良好的应用潜力。为了提高getPCR中indel敏感引物区分编辑序列的能力,我们通过半理性分子进化的方式对Taq酶的特异性进行改进,获得了一种带有S577A、W645R和I707V三个氨基酸替换的高特异性Taq DNA聚合酶变体。应用此高特异性Taq DNA聚合酶进行getPCR分析时,不管是检测indel还是单碱基变异特异性均大幅提高,完全消除了来自错配模板的扩增信号,大大提高了定量的准确性。进一步,我们结合数字PCR(dPCR)在基因检测绝对定量方面的优势,在dPCR平台上通过getPCR方法检测基因编辑和基因变异。我们发现,与现有的采用荧光探针区别基因编辑序列和野生序列的GEF-dPCR方法相比,我们的基于高特异性Taq DNA聚合酶的get-dPCR方法具有更强的鉴别基因变异的能力,在indel以及单碱基变异的检测中,可完全区分野生序列和发生变异的序列,不仅实现了精准检测,还解决了 GEF-dPCR方法中因为荧光探针从错配模板也能产生非特异信号带来“雨点”的困扰。这表明我们的get-dPCR方法在评估基因编辑以及单碱基DNA变异方面均有优越的准确性。更多还原

【Abstract】 Cancer has become the main cause of human death worldwide.As a complex disease,excavating,screening and annotating cancer-related genetic variation sites are of great significance for the prediction,diagnosis and treatment of cancer.A large number of genetic variation sites that are significantly associated with cancer risk have been discovered through genome-wide association analysis(GWAS).However,the risk variation loci discovered by GWAS are usually common mutations with a variation frequency greater than 1%,excluding rare mutation loci that may have greater genetic effects.Furthermore,whether these risk variant sites discovered by GWAS are causal sites that lead to disease risk still requires a lot of functional analysis and screening work.A large number of studies have shown that most risk variant sites mainly affect downstream gene expression by changing the binding of specific transcription factors,thereby leading to disease risk.Therefore,this project starts from the transcription factors that are closely related to the risk of occurrence and development of cancer.By analyzing the ChIP-seq data of tumor clinical samples,we discover the mutation sites that affect the binding of transcription factors and systematically explore regulatory mutation sites that affect key transcription factor binding.HOXB13 transcription factor plays an important regulatory function in prostate cancer.Previous reports have shown that the prostate cancer risk SNP site rs339331 mediates HOXB13 to regulate the expression of the downstream target gene RFX6,thereby affecting cancer risk.This study focused on the regulatory role of HOXB13 transcription factor and comprehensively screened SNP sites that mediate the regulatory role of HOXB13.We obtained HOXB13 ChIP-seq data from 42 clinical samples of prostate patients,and analyzed all SNP sites present in the HOXB13 chromatin binding peak.Then we evaluated the differences between alleles for binding ability to HOXB13 to screen mutation sites that can affect HOXB13 binding.Furthermore,combined with the RNA-seq data of HOXB13 knockdown cells,4814 potential HOXB13-mediated gene regulatory SNPs were obtained based on changes in adjacent genes or interacting genes.Subsequently,the regulatory activities of 9628 alleles of these 4814 SNPs were evaluated through massive parallel reporter analysis(MPRA)experiments,and 190 SNP sites with regulatory activity and allelic differences were obtained.These sites may play an important role in the occurrence of the disease by affecting the regulatory reprogramming of the HOXB13 gene in prostate cancer.In addition,42.6%of the SNVs among these 190 mutation sites have a mutation frequency of less than 1%in the population.Therefore,these variants with gene regulatory functions discovered in our research work can complement the risk sites discovered by GWAS analysis,laying a good foundation for the comprehensive discovery and elucidation of the molecular mechanisms of cancer genetic susceptibility risks.Genetic variation plays an important role in human disease risk.It is of great significance to interpret the function of genetic variation and elucidate its pathogenic mechanism.Gene editing is commonly used to construct genetic variation in research.At present,CRISPR/Cas9 technology has been widely used for targeted gene editing in various organisms.However,how to accurately detect genetic variations introduced by gene editing is still a problem that needs to be solved.This project developed a rapid and accurate method for evaluating gene editing based on qPCR technology,called getPCR(genome editing test PCR).This method takes advantage of the sensitivity of Taq DNA polymerase to nucleotide mismatches at the 3’end of the primer during the initiation of DNA replication.It uses a primer that spans the Cas9 cleavage site with 3-5 3’ end bases to sense and discriminate the indel sequences.By this,it can selectively amplify and quantify the proportion of wild-type sequences,thereby achieving a quantitative assessment of gene editing.This method can not only accurately detect random indel mutations introduced by NHEJ,but also can accurately quantify the base variations introduced by HDR or base editor methods using primers matching the anticipated target sequences.They have good application potential in gene editing variation evaluation and genotyping of cell clones.In order to improve the ability of indel-sensitive primers to distinguish edited sequences in getPCR,we improved the specificity of the Taq polymerase through semi-rational molecular evolution and obtained a highly specific Taq with three amino acid substitutions of S577A,W645R and 1707V.When this highly specific Taq DNA polymerase is used for getPCR analysis,the specificity for detecting indel or single-base variations is greatly improved,completely eliminating amplification signals from mismatched templates,and greatly improving the accuracy of quantification.Furthermore,we combined the advantages of digital PCR(dPCR)in absolute quantification of gene detection to detect gene editing and genetic variation through the getPCR method on the dPCR platform.We found that compared with the existing GEF-dPCR method that uses fluorescent probes to distinguish between gene-edited sequences and wild-type sequences,our get-dPCR method based on highly specific Taq DNA polymerase has a stronger ability to identify genetic variants.In the detection of indel and single base variations,this method can completely distinguish between wild sequences and mutated sequences.This not only allows for accurate detection,but also solves the problem of non-specific signals of fluorescent probes from mismatched templates,also known as "raindrops" problem in the GEF-dPCR method.This demonstrates the superior accuracy of our get-dPCR method in assessing both gene editing and single-base DNA variation.更多还原