Application Research Of CRISPR/dCas9 System Combined With Electrochemical Technology For Single Nucleotide Variation Non-amplified Detection Method

Purpose:Malignant tumor is a serious threat to human health.In recent years,the morbidity and mortality of malignant tumor have shown a trend of continuous increase.More rapid and accurate diagnostic techniques are urgently needed to guide targeted therapy.Compared with traditional radiotherapy and chemotherapy,targeted therapy has the advantages of strong specificity and low toxicity.At present,the targeting of drug therapy has been gradually improved,which has significant efficacy for a variety of malignant tumors.Different medication strategies corresponding to specific gene mutations have been gradually popularized in clinical practice.How to detect gene mutation quickly and easily is the key to prompt treatment of patients.Traditional methods for single-nucleotide variants based on the amplification and the fluorescent signals require expensive reagents and cumbersome instruments,and they are time-consuming for each trial.Patients often need to go to large provincial hospitals for sampling and testing,and the samples will be sent to special medical inspection institutions for sequencing and bioinformatics analysis.It takes about 20days to get the test results,and medical accessibility is difficult to be satisfied.In this paper,a sensor chip based on porous anodic alumina（PAA）was developed to carry out the concomitant diagnosis of medical and health care and various nucleic acid related diseases based on pharmacy.The chip was modified in several steps,and then modified by the deactivatedCas9（dCas9）protein and the guide RNA（sg RNA）specifically binding to the targeted sequence.Finally,the electrochemistry ion current rectification（ICR）principle was used to measure the electrical signal.The system that was developed for the first time was called CRISPR-PAA.Method:The detection principle of CRISPR-PAA platform is based on the multi-step surface modification of PAA chip binding dCas9 protein of CRISPR/dCas9 system,specific sg RNA and target sequence including the whole genome of single-base mutation clinical samples or virus.Using the principle of ICR,the nonlinear current potential（I-V）curve is obtained due to asymmetric ion migration by electrochemical technology,and the electrical signal is detected for the modified PAA chip.The change of electrical signal is due to the combination of nucleic acid chip and negative charge,and the electrode in the buffer will cause the change of K⁺ion transmission efficiency and potential on both sides of the chip,and the detected current value is significantly enhanced,which can intuitively and efficiently reflect the state of the combined target sequence,so as to achieve the purpose of specific detection and real-time diagnosis.The first part of the study is to prepare dCas9 protein whose concentration and purity meet the requirements of the detection platform.By constructing dCas9 protein expression vector,small-scale expression screening suitable expression conditions,pilot-scale fermentation,expansion,cultivation and purification of the protein,and verifying whether the protein-related parameters meet the standards.In the second part,the preparation procedure of multifunctional PAA chip is studied.After heat treatment of high-purity aluminum sheet at appropriate temperature,uniform Al₂O₃ porous film is obtained by two-step anodic oxidation operation and film removal polishing in acidic solution.The third part of the research is the construction and verification of the nucleic acid detection platform.The modified PAA chip is characterized by SEM electron microscopy and X-ray photoelectron spectroscopy（XPS）,and the optimal electrode buffer concentration and p H value used in the system are screened.The systematic stability of the E.coli standard sequence and the SARS-Co V-2 S,N,and ORF1ab genes were verified.The fourth part of the study is to verify the detection of single-base mutations in clinical samples.Through the extraction of whole-genome DNA from surgical samples of patients with large B-cell lymphoma,the specific sg RNA was designed for single-base mutation MYD88^L265P,and the binding activity was determined and compared with traditional detection.q RT-PCR and Sanger sequencing further verified the stability and practicability of the CRISPR-PAA electrochemical nucleic acid detection platform.Results:A CRISPR-PAA electrochemical nucleic acid detection platform was developed and verified.A CRISPR-PAA electrochemical nucleic acid detection platform was successfully developed and verified.The binding efficiency of standard sequence of E.coli,SARS-Co V-2 related genes and whole genome sequence of tumor single base mutation samples can be directly reflected by the change of K⁺ion transport efficiency and potential in electrode buffer.dCas9 protein with high purity and concentration suitable for detection platform was obtained,and the surface modification effect of PAA chip was successfully verified,and satisfactory detection effect was obtained for different targeting sequences.In the first part of the study,the E.coli expression vector for dCas9 protein was successfully constructed,and the optimal expression conditions obtained from the small-scale expression screening were:IPTG concentration:1 m M;induction time:8h;induction temperature:16℃.After fermentation and purification,the protein concentration reached 1.34 mg/m L,and the purity was 98.1%.In the second part of the study,the etching operation was carried out in a phosphoric acid/chromic acid mixed acid solution through two-step electrochemical anodization,and the Al and the ordered pits on it were completely preserved,which greatly simplified the preparation process.The effective sensing area is determined on a circular membrane with a diameter of 0.5 mm,and finally a modifiable PAA chip suitable for clinical nucleic acid detection is obtained.In the third part of the study,the XPS method and SEM electron microscope were used to characterize the modified PAA chip,confirming that the element changes after each step of modification proved the stability and feasibility of the modification method.Scanning electron microscopy was used to compare the morphological changes of blank chip and dCas9 protein modification.The top view and cross-sectional view show that the protein and the chip are well combined.It is determined that the electrolyte KCl concentration is 5m M,and it is more stable at p H=7.6.The detection and verification results of the system against the E.coli standard sequence and the SARS-Co V-2 S gene,N gene and ORF1ab gene are good.First,the binding activity of the system is verified by the DNA binding reaction,and the DNA concentration gradient is set to compare the current value.The detection current can reach more than 250μA,which shows that the system is sensitive and efficient.In the fourth part of the study,specific primers and sg RNA were designed for the whole genome DNA of clinical samples and the mutation site of MYD88^L265P,and the specific identification of MYD88^L265P and the verification of sg RNA activity were completed.The limit of detection was calculated to be 26.3 f M,which proved that the detection platform has high sensitivity.Compared with traditional q PCR and Sanger sequencing methods,the specificity and practicality of the CRISPR-PAA electrochemical system have been further verified.Conclusion:A CRISPR-PAA electrochemical nucleic acid detection platform was developed and verified by combining CRISPR/dCas9 technology and electrochemical detection method.The detection of known sequence,SARS-Co V-2 gene and single-base mutation sequence in clinical samples further demonstrates the effectiveness and practicability of the platform,which will provide an easy-to-operate,specific,efficient solution for the diagnosis and immediate treatment of genomic or tumor SNV.