Abstract

THIS study is based on a“Collateral cleavage” crispr-Cas detection approach that targets binding to sgrnas to activate CAS enzymes (CAS13A1 and CAS14A1) , it is difficult to distinguish the source of detection signal and avoid the interference of the mutant nucleic acid fragment when the target system contains the target mutant nucleic acid fragment (1 base is different) , thus, the problem of false positives is todesign a protocol, through the sequence design and length screening of Pna 'shackle' chain, accurate and effective 'blocking' target mutation nucleic acid fragments, therefore, we can improve the detection of target specificity and achieve the ability of single-base recognition detection. This scheme is an improvement on the specificity of CRISPR/Cas (Cas13 and CAS14) nucleic acid detection methods.

It is difficult to distinguish target RNA from mutant RNA.

Coexisting target RNA and mutant RNA in the reaction system.

The design of the shackles.

The mutant RNA for each single base pair was shackled with a pre-designed PNA.

CAS13A protein and CSM6 protein were linked in tandem to improve the detection efficiency, and the problem of base mismatch was solved by PNA shackle, which made the detection accurate to single-base recognition, fluorescent protein was used as the reporter signal. On this basis, we also develop a hardware to get the detection results.

Now it is possible to distinguish between target RNA and mutant RNA!