Research On Killing Cancer Cells With CRISPR Targeting Genomic Repeat Sequences Alu

Cancer,as one of the major diseases threatening human health,has the characteristics of being difficult to detect early and difficult to treat in the later stages,causing great harm to patients’ physical and mental health.Although some progress has been made in recent years in areas such as surgical treatment,radiotherapy,targeted therapy,and immunotherapy,the treatment effects still fall far short of meeting patient needs.Therefore,continuous exploration and innovation of cancer treatment methods remain a daunting task.The essential cause of cancer is mutations in oncogenes or tumor suppressor genes,highlighting the importance of genetic approaches for cancer treatment.The CRISPR gene editing system has been widely used in the study of oncogenes and tumor suppressor genes,the construction of cancer models,and cancer diagnosis.However,current research on the direct killing of cancer cells using CRISPR is relatively limited.Even in these limited studies,the focus is primarily on using CRISPR to disrupt exon regions that encode functional proteins in cancer cells.The effectiveness of this killing strategy is limited by CRISPR editing efficiency and cancer heterogeneity.There are several difficulties in studying the direct killing of cancer cells using CRISPR: avoiding editing of normal cells while editing cancer cells;efficiently delivering the CRISPR system to cancer cells;and enhancing the editing efficiency of the CRISPR system inside cells.This article focuses on using CRISPR to directly kill tumor cells,aiming to solve the aforementioned problems and provide new ideas and methods for cancer treatment.In this study,we targeted the largest non-coding repeat sequence in the human genome,Alu sequences,and used CRISPR to cleave Alu repeat sequences widely distributed in cancer cell genomes.This approach generates extensive chromosomal breaks in cancer cells,fundamentally disrupting the DNA of cancer cell genomes and ultimately killing cancer cells.This study provides a new strategy for exploring novel cancer treatment methods.This paper mainly conducts research in the following four aspects:1.Screening and Identification of Consensus Alu SequencesAlu sequences are important repetitive sequences in the human genome,which have been subdivided into multiple subfamilies due to the long evolutionary history of humans.There are some differences in the consensus sequences of these subfamilies,so it is necessary to select an appropriate Alu consensus sequence as the target of the CRISPR system.To find a suitable Alu consensus sequence,this study used the BLAST method to search the distribution of Alu consensus sequences from different families in the human genome GRCh38.p14.Experimental results showed that the Alu consensus sequence obtained from the position frequency matrix is widely distributed on every chromosome of human cells,and is more suitable as a CRISPR cleavage target for destroying genomic DNA compared to other Alu family consensus sequences.2.Damage to Genomic DNA by Extracellular Cleavage of Alu Consensus SequencesTo explore whether the CRISPR system can cleave Alu consensus sequences and damage genomic DNA,we conducted extracellular cleavage experiments.In the experiment,we used the Cas12 a nuclease and a target cr RNA specific for the Alu sequence to cleave Alu consensus sequence DNA fragments,Alu consensus sequence PCR amplification products,HEK-293 T cell genomic DNA,five different cancer cell genomic DNAs,and genomic DNAs from five different organisms.At the same time,we compared the damage to genomic DNA caused by cleavage of Alu consensus sequences with that caused by cleavage of single-copy genes.The experimental results showed that CRISPR can cleave Alu consensus sequence DNA fragments and also cleave amplified Alu sequences from cancer cells.Additionally,we found that CRISPR cleavage of Alu consensus sequences leads to complete degradation of human genomic DNA and can completely digest genomic DNA from all five cancer cell lines.However,this cleavage damage is limited to human genomic DNA containing Alu sequences,while cleavage of singlecopy genes has little effect on genomic DNA integrity.The results demonstrate that the CRISPR system can cleave Alu consensus sequences and damage genomic DNA,with this damage being primarily confined to human genomic DNA containing Alu sequences.3.Evaluation of Intracellular Cleavage of Alu Consensus Sequences on Toxicity in Cancer CellsTo investigate the intracellular cleavage of Alu consensus sequences and the resulting cytotoxicity in cancer cells,we cloned the CRISPR system that cuts Alu sequences into plasmids integrated with a green fluorescent protein(GFP)reporter system,and transfected the plasmids into cells such as HEK-293 T using Lipofectamine 3000.We evaluated the transfected cells using fluorescence microscopy,flow cytometry,and cell toxicity assays to investigate the physiological status of cells after cleavage of Alu sequences.Additionally,we characterized the DNA double-stranded breaks using immunofluorescence staining.To enhance the delivery level of the CRISPR system,we packaged the CRISPR system that cuts Alu sequences into recombinant adenovirus Ad5.The experimental results showed that traditional methods of editing single-copy genes may allow a small number of cells to survive the adverse effects of gene editing and then proliferate again,while CRISPR cleavage of Alu repeat sequences makes it difficult for cells to survive and continue proliferating.Additionally,we found that cleavage of different Alu consensus sequences or use of different Cas enzymes can result in different cell toxicities.Immunofluorescence analysis showed that cleavage of Alu consensus sequences causes massive DNA double-stranded breaks in cancer cells.Delivery of the CRISPR system using adenovirus as a vector increases transduction efficiency.Furthermore,we packaged the modules expressing Cas12 a protein and cr RNA into recombinant Ad5 adenovirus for transduction,which not only reduces biosafety risks but also retains co-infection efficiency and cell mortality rates.These experimental results demonstrate that intracellular cleavage of Alu consensus sequences can destroy genomic DNA and produce significant cell toxicity.4.The Design and Optimization of a CRISPR System for Specific Cleavage of Alu Consensus Sequences in Cancer CellsTo specifically target cancer cells for Alu sequence cleavage,this study proposes a CRISPR editing system based on the Alpha-Fetoprotein(AFP)promoter and site-specific recombinase,and optimizes its design.The AFP promoter is a tumor-specific promoter,and this study uses AFP-positive liver cancer cell lines as the research object,comparing the expression efficiency of CRISPR-Cas12 a driven by different structures of the AFP promoter,and selects the optimal AFP promoter p AFP(a2b)for subsequent experiments.By combining site-specific recombinase and tandem terminator elements,multiple models for specific expression of the CRISPR system in liver cancer cells were constructed,and their specificity and killing efficiency were evaluated.Experimental results demonstrate the successful construction of a dual recombinant adenovirus system with both specific expression and high expression efficiency,achieving targeted killing of various AFP-positive liver cancer cells.The dual recombinant adenovirus system based on the m CMV promoter,AFP promoter,site-specific recombinase,and tandem terminator elements maintains expression specificity comparable to a system using the pure AFP promoter,and achieves an expression efficiency of 74.5% that of the pure m CMV promoter system.This research breaks the previous framework of CRISPR systems,which were limited to editing exon gene sites to eliminate cancer cells.The experimental results show that compared to single-copy genes,cleaving Alu repeat sequences can more effectively improve the efficiency of CRISPR in killing cancer cells.In terms of delivery methods,this study used a dual-recombinant adenovirus system,which not only ensures safety but also significantly improves transduction efficiency,surpassing liposome transfection methods.Furthermore,this study proposes an innovative framework that uses tumor-specific promoters and site-specific recombinases together,resulting in significant improvements in both expression efficiency and specificity.In conclusion,this research provides a new innovative strategy for treating cancer at the genetic level,which brings new hope for cancer treatment through efficient disruption of cancer cell genomes.