Research Of CRISPR-Cas12b Trans-cleavage Activity And The Development Of Cas12b-assisted Nucleic Acid Detection Platform

Due to the high sensitivity,specificity and simplicity,CRISPR based nucleic acid detection methods have shown great promise in clinical diagnostics and have drawn much attention.The principle of these methods is based on the trans-cleavage activities of some Cas proteins,including the DNA-targeting and ssDNA trans-cleaving Cas12a.Combined with amplification,Cas12a has been employed to develop HOLMES(one HOur Low-cost Multipurpose highly Efficient System)or DETECTR(DNA Endonuclease Targeted CRISPR Trans Reporter).However,the amplification and detection processes are separated in both systems.Here,we did further research on Cas12b,a Class 2 Type V CRISPR effector like Cas12a which also have trans-cleavage activity.We found both dsDNA and ssDNA targets could trigger Cas 12b trans-activity and Cas12b showed higher activity with ssDNA.Next,we test temperatures required for its trans-activity and found the suitable temperature ranged from 45? to 55? with dsDNA target and 35? to 65? with ssDNA target.Then,we determined whether PAM is required for Cas12b trans-cleavage and the lowest target concentration for triggering when combined with Loop-Mediated Isothermal Amplification(LAMP)or not.Furthermore,we also test the ability to distinguish single nucleotide polymoiphisms(SNPs)of Cas12b and found single-nucleotide mismatch can be recognized for both dsDNA and ssDNA targets with full length or truncated sgRNA.Thus,Cas12b trans activity is of both high sensitivity and high specificity.Because both LAMP amplification and Cas12b trans cleavage have a wide temperature range,we wondered if we could integrating these two steps in one reaction system.The integrated system was tested under 48? to 60?.We found the signal was hardly detectable when the temperature was above 57.8?.Then,temperature of 55? was finally selected for this one-step reaction and we achieved target DNA quantification successfully which concentration ranging from 10-5 nM to 1 nM.The one step reaction system combined Cas12b trans cleavage and LAMP amplification was termed HOLMESv2.Besides,using DNA polymerases which contained the activity with either DNA or RNA templates to amplify RNA target directly,it can be more robust and convenient for RNA detection with HOLMESv2.