A Universal CRISPR/Cas12a DNA Sensing Platform Based On Proximity Extension And Transcription-unleashed Self-supply CrRNA

Objective: To establish a universal and new method of detecting nucleic acids based on the CRISPR/Cas12 a,but the target nucleic acids we wanted to detect didn’t require containing TTTN and didn’t need to multiply design crRNA,which can achieve rapid,simple and sensitive detection.Methods: We designed two kinds of probes including extension probe and template probe.When the target DNA was present,the target DNA was recognized and bound to the two adjacent probes,and then the adjacent binding and extension were trigged,extending the promoter sequence of T7 RNA polymerase.By identified its specific promoter,T7 RNA polymerase initiated transcriptional amplification to produce a large number of single-stranded crRNA products.Subsequently,the ds DNA probe containing PAM sequence and Cas12 a protein were added to the above buffer solution to assemble a triple complex,which could activate the trans-cleavage activity of Cas12 a,and continuously cleavage the ss DNA reporter probe labeled with the fluorescence group of FAM and the quenching group of BHQ-1,thus generating a strong fluorescence signal.Non-denatured polyacrylamide gel electrophoresis and fluorescence intensity were performed to verified the process.Results:(1)According to the fluorescence spectrum,we could see that the positive sample had a very strong fluorescence signal while the negative sample only has a very small fluorescence signal.According to the PAGE electrophoresis,we could see the transcribed RNA bands and the disappear ssDNA.Above results showed the trans-cleavage activity of Cas12 a and the feasibility of the whole experimental scheme.(2)We optimized a series of experimental conditions.The optimal conditions were determined as follows: the number of complementary bases between the extension probe and template probe was 6,the concentration of T7 RNA polymerase was 30 U,the time of transcription amplification was 3 hours,the concentration of Cas12 a was 50 n M and the time of Cas12 a trans-cleavage of single stranded DNA was60 minutes.(3)We carried out the experimental performance analysis under the optimal experimental conditions and obtained a linear equation F=22.373 l C-30.270 with a correlation coefficient of 0.995 at 500 fM～100 pM.(4)We also analyzed the specificity,repeatability,generality and anti-interference ability of the experimental method,and the results showed that the method had excellent specificity,repeatability,generality and anti-interference ability.Conclusion: We established a universal CRISPR/Cas12 a DNA sensing platform based on proximity extension and transcription-unleashed self-supply crRNA to realize a rapid,simple and sensitive strategy for detection of nucleic acids.This method has a good repeatability,specificity and anti-interference.In addition,it also has the following advantages;(1)It gets rid of the defect that Cas12 a can only recognize target nucleic acid containing PAM sequence;(2)It is not necessary to design crRNA multiple times to activate Cas12a;(3)It can be used for general detection of a wide range of DNA/RNA sequences,which broadens the application of CAS12 a system in the field of nucleic acid detection.