New Methods And Performance Study For Detecting Breast Tumor Markers Based On CRISPR/Cas12a System

By 2020,breast cancer has become the most common cancer in the world.The detection of tumor markers is of great significance for early diagnosis,real-time monitoring,chemotherapy resistance and prognosis evaluation of breast cancer.However,the traditional detection methods have some limitations,and it is difficult to achieve rapid and immediate detection.As an emerging technology,CRISPR/Cas technology is considered to be the core of the next generation of diagnostic technology.Biosensors constructed by CRISPR/Cas technology have the advantages of high sensitivity and specificity,and have great potential in real-time detection.Therefore,the CRISPR/Cas12a system was introduced into the detection of three different types of breast cancer tumor markers(telomerase,PIK3CA^H1047R,miR-21)in this paper,aiming to provide new methods and ideas for the construction of a more sensitive and convenient breast cancer tumor marker sensing platform,and also expand application fields for the CRISPR/Cas system.The main research results are as follows:（1）Here,a sensor that telomerase extended activators to unlock the ssDNase activity of CRISPR/Cas12a was created to detect the telomerase activity,realizing the ultra-sensitive and real-time detection of telomerase activity in MCF-7 cells.Under the action of telomerase,the primers continue to extend,and the amplified fragments can be recognized by crRNA,thus activating the trans-cleaping activity of Cas12a,which is equivalent to the realization of double amplification of signals.Based on the fluorescence or lateral flow assay,we achieved the ultrasensitive and point-of-care detection of telomerase activity low to 57 cells/mL and 5.7×10² cells/mL in about one hour,respectively.In addition,the sensor also had potential in the classification of breast cancer cell subtypes.The established fluorescence detection and paper-based POCT platform were highly sensitive,simple and quick,which provided a new method and idea for the detection of telomerase activity.（2）Here,a fluorescent biosensor which combines cascade strand displacement amplification（C-SDA）and CRISPR/Cas12a was established to ultra-sensitively detect gene-PIK3CA^H1047Rmutation.The mutated gene-PIK3CA^H1047Rcan combine with complementary sequence to form an intact recognition site for endonuclease FspI.In the presence of FspI,mutated gene-PIK3CA^H1047R can be cleaved at the mutation site to produce a DNA fragment that can induce SDA or C-SDA.Compared with the conventional SDA,the improved SDA in this paper designed one more Nt.BbvCI site in its DNA template,and realized cascade amplification,which greatly improved its amplification efficiency.Through C-SDA,a large amount of single-stranded DNA which acts as activators to motivate the trans-cleavage ability of CRISPR/Cas12a system was obtained.Under optimized conditions,we achieved the ultrasensitive detection of gene-PIK3CA^H1047R mutation low to 0.001%.Besides,the biosensor performed well in the labeled recovery experiment of human serum samples,indicating it owns potential in detecting low-abundance gene-PIK3CA^H1047R mutation.（3）Here,a biosensor platform based on multiple cascade strand displacement amplification（MC-SDA）and CRISPR/Cas12a for sensitive miR-21 fluorescence detection was proposed.The target can act as a primer to trigger the nucleic acid amplification processof C-SDA.On this basis,a new template was introduced,and the intermediate product of C-SDA could combine with the added template to trigger another SDA cycle amplification process,which realized multiple cascade amplification.The improved MC-SDA method showed a higher amplification efficiency.MC-SDA products acted as activators to unlock the trans-cleavage activity of CRISPR/Cas12a,and the specificity and signal amplification characteristics of CRISPR/Cas12a system further improved the performance of the sensor.Based on MC-SDA-CRISPR/Cas12a,the actual detection limit was 10 fM.In addition,the biosensor showed a good performance in selectivity and human serum sample experiments,indicating a certain practical application potential.