Research On Novel Methods Of Gene Transcriptional Activation And DNA Targeted Enrichment Based On CRISPR/dCas9 Technology

Based on the CRISPR/dCas9 technique,this study added a 24 bp capture sequence at the 3’ end of the sgRNA to form a kind of capture sgRNA(csgRNA).After forming a complex with dCas9,the csgRNA can direct dCas9 to the target sequence.Using the base complementary principle,the capture sequence on the dCas9-csgRNA complex interacts with a linear DNA fragment with a complementary single strand,thus achieving the following experimental objectives:(1)anchored a linear CMV fragment with complementary single strand to the promoter region of the target gene,allowed the target gene to be transactivated to achieve transcriptional activation,so that the expression level of the target gene is increased to achieve a specific cell biology objective;(2)Targeted the dCas9-csgRNA complex to the target DNA sequence in vitro,followed by enrichment of the target DNA with magnetic beads with complementary single-stranded linear DNA fragments,which were then constructed the library and sequenced;(3)Targeted the intracellularly expressed dCas9-csgRNA complexes to the DNA of interest(promoter),followed by enrichment of the DNA/protein complexes with magnetic beads with complementary single-stranded linear DNA fragments,which were then analyzed to identify regulatory sequences and transcriptional regulators involved in the regulation of the gene of interest.The research content of this thesis is divided into the following three parts: 1.Transactivation of target genes using trans enhancersThe blunt CMV(blunt CMV,bCMV)sequence was amplified from the pEGFP-N1 vector,and then a single-chain overhang sequence complementary to the csgRNA capture sequence was formed at the 3’ or 5’ end of the CMV sequence using a nicking enzyme.A sticky-end CMV sequence(stick CMV,sCMV)was thus prepared.After the CRISPR/dCas9 and csgRNA expression vectors were co-transfected with the sCMV fragment,the expressed CRISPR/dCas9-csgRNA complex could recognize and bind the target sequence.Finally,the sCMV was anchored to the target sequence(promoter region of target gene)by annealing with the capture sequence of csgRNA.The sCMV sequence was thus recruited to the promoter of the target gene.Depending on the high transcriptional activation activity of CMV promoter,the transcription of target gene was highly activated by trans enhancer.In this study,the endogenous HNF4α gene was transcriptionally activated in 7 types of cells(293T,A549,SKOV3,HT29,HepG2,PANC-1,and HeLa).The experimental results showed that the trans enhancer highly activated the transcription of HNF4α gene in the above 7 types of cells.The transcriptional efficiency of HNF4α gene was more than 10 times.In this study,the other 9 genes were selected as target genes(TCF3,ASCL1,Ngn2,Oct4,Nanog,TNFAIP3,CASP9,CSF3,and Sox2)for transcriptional activation.Using the trans enhancer method to activate target gene transcription and expression in the above 7 cells,the experimental results show that the transcription of the above 10 genes can be up-regulated to different degrees;however,the fold change of the up-regulated expression of the target gene in different cells has cell-specificity.The reason for this difference may be the degree of openness of these genes in the above cell lines.The transactivation of HNF4α and TCF3 genes in HepG2 cells and PANC-1 cells,respectively,revealed that HepG2 and PANC-1 cells showed the following changes:(1)Downregulation of tumor marker gene expression;(2)Changes in cell cycle,in which cells in G0/G1 phase were significantly increased;(3)Functional genes in normal cells were upregulated in different degrees;(4)Cell migration and division and proliferation were decreased.The above results indicate that cancer cells can be transformed and differentiated by upregulating key genes in tumor cells.Trans enhancer provides a new method for the implementation of such cancer therapy.The transcription factor as the core of the intracellular transcriptional regulatory network will be the key gene of choice for this type of cancer therapy.Compared with the previous method of activating the transcriptional expression of the target gene by means of dCas9,the trans enhancer technology used in this study can achieve more efficient activation.Trans enhancer technology provides new method and approach for the target selection and verification of medical treatment and other aspects.2.Targeted enrichment of genomic target sequencesIn this study,using the dCas9-csgRNA complex to targetedly enrich the target DNA region in vitro,the following results were achieved:(1)multiple csgRNAs can be used together after mixing to enrich multiple target DNA regions in high-throughput format;(2)Targeted enrichment of the mutated sequence from the mixed sample.Targetedly enriched DNA can be used to conveniently construct the library and sequenced for SNP analysis.In this study,six genes(APOE,AKT1,TP53,IL6,BRCA1,and TERT)were selected and 54 csgRNAs were used to target and enrich the exon regions of the six genes.The technology includes the following steps:(1)Extracting genomic DNA and digesting the genomic DNA with Tn5(tagmentation),recovering 300–1000 bp target sequences;(2)Incubating the genomic DNA fragment with the dCas9-csgRNA complex for 1 h,and the dCas9-csgRNA-target DNA complex was enriched with magnetic beads coupled with a complementary single-stranded linear DNA fragment;(3)analyzing the enriched target sequence with clone sequencing or nextgeneration sequencing(NGS);(4)bioinformatic analysis of reads.After the above operations,specific recognition and enrichment of the target region can be achieved.Through the complete pairing of the dCas9-sgRNA complex with the target sequence,the interference of similar family genes and homologous genes in the sample can be excluded,allowing the true targets to be precisely enriched.In this study,different amounts of csgRNA were mixed and used for the enrichment of multi-target DNA sequences.It was verified by experiments that mixing 10 or 54 csgRNAs had no effect on the enrichment effect of target fragments.Further,by changing the target sequence of the csgRNA so that it is more inclined to recognize and bind to the mutated DNA sequence,the csgRNA can specifically enrich the mutated sequence from the mixed sample and exclude the normal sequence.This method will help to detect pathogenic mutations in an early and timely manner and strive for more time for diagnosis and treatment of such diseases.This method does not rely on the most widely used nucleic acid hybridization method or PCR amplification method for the enrichment of the target sequence.The specific capture of the target sequence is dependent on the specific interaction of the CRISPR/dCas9-csgRNA complex and the target DNA,which is achieved in a normal-temperature,non-denaturing,mild gripping technique that is very beneficial to avoid non-specific noise artifacts from nucleic acid hybridization methods or PCR amplification methods.It can be seen that this study provides a powerful new technology for the targeted enrichment of DNA.3.Targeted enrichment of regulatory sequences and transcriptional regulatory proteins of target genesThis study describes the use of dCas9 and csgRNA expression vectors to express and assemble the dCas9-csgRNA complex in cells,which recognizes and binds to the promoter region of the target gene.The csgRNA capture sequence was then used to enrich and identify the remote regulatory sequences,proximal regulatory sequences and transcriptional regulatory factor proteins bound to these sequences of the target gene.The nucleic acid sequences are enriched,and a sequencing library is constructed using a Tn5 transposome for high-throughput sequencing and analysis,while protein samples are analyzed with mass spectrometry that uses the Label-free method to qualitatively identify and quantify protein species in samples.The entire enrichment process includes the following five steps:(1)transfection of dCas9 and csgRNA expression vectors and the expressed dCas9-csgRNA complex targets to the promoter region of the target gene;(2)formaldehyde cross-linking cells and separation of cell nuclei;(3)Fragmentation of formaldehyde-crosslinked chromatin using Tn5 transposomes;(4)Isolation and enrichment of protein-chromatin complex containing csgRNA using magnetic beads with complementary single-stranded linear DNA fragments;(5)After the decross-linking of the enriched products,the nucleic acid samples were subjected to high-throughput sequencing analysis and protein samples were analyzed by mass spectrometry.Using this method,we analyzed the regulatory elements of five target genes(HBB,MYC,HNF4α,TCF3,and RelA)in three types of cells,obtaining more precise enhancer regulatory elements.The method does not rely on antibodies,is simple and low-cost,only needs to build a csgRNA expression vector,which can be used to analyze the regulatory model of the target gene.This study thus provides a new method for studying the transcriptional regulatory mechnism of the target gene.This study has for the first time developed a new technique that allows for the rapid identification of target gene regulatory regions.