Cas Molecular Switches Built On Anti-CRISPR Proteins Their Biosensing Applications

CRISPR(clustered regularly interspaced short palindromic repeats)and CRISPR-associated proteins(Cas)constitute a nucleic acid-based adaptive immune system in bacteria and archaea that uses RNA-guided nucleases to cleave invading nucleic acids.Notalbly,Cas12a and Cas13a,which belong to the class 2 CRISPR-Cas system,can continuously cut single-stranded nucleic acid(trans-cleavage)after recognizing and cleaving the target sequence(cis-cleavage)and are used to develop the next generation of molecular diagnostics.In addition,using functional nucleic acids as signal transducers,the application scope of the CRISPR-Cas biosensing has also been extended to non-nucleic acid analytes,such as small molecules and proteins.However,incompatibility issues between Cas’s trans-cleavage and functional nucleic acid elements may interfere with the detection,thus requiring complex front-end design or multi-step reactions.Anti-CRISPR protein(Acr)derived from phage and prophage can inhibit the function of the CRISPR-Cas system,protect phage and prophage from the degradation of the CRISPR-Cas system,providing the basic elements for constructing responsive CRISPR-Cas systems.In this paper,on the basis of the systematic analysis of the interaction between Acr and CRISPR-Cas,we designed and constructed two types of responsive CRISPR-Cas12a systems and developed a new CRISPR-based biosensing method and protease-responsive gene-editing system.The specific research contents are as follows:1.Design and construction of Cas12a in response to histone deacetylase(HDAC)activity.AcrVA5 has acetylase activity,which can catalyze the acetylation of the Lys595 site of Cas12a and inhibit its activity by affecting the binding of target DNA.In this chapter,we used deep learning and protein-protein docking to predict whether HDACs could catalyze the deacetylation of acetylated Cas12a(Ac-Cas12a).Taking sirtuin-1(Sirt 1),a widely studied histone deacetylase,as an example,we made the prediction that the active center of Sirt 1 could access Ac-Lys595 and catalyze its deacetylation.The experimental results showed that Sirt 1 can activate both the cis-and trans-cleavage activities of LbCas12a in the presence of NAD~+.We further constructed a simple and rapid preparation method of Ac-Cas12a,which laid a foundation for the development of a CRSIRP-based system for HDAC activity assay.2.Ac-Cas12a-based method for the detection of HDAC activity.In this chapter,we constructed a sensitive method for the bioanalysis of HDAC activity by utilizing the high-efficiency signal amplification mediated by the trans-cleavage activity of Cas12a.Under the optimized buffer conditions,we realized the compatibility between the deacetylation reaction catalyzed by HDAC and the trans-cleavage reaction of Cas12a,achieving one-pot rapid detection of deacetylase activity based on Ac-Cas12a.The method showed a linear response to Sirt 1 in the concentration range of 0.5–100 nM with a detection limit of 0.2nM,which is 1/50 of the traditional two-step fluorogenic substrate peptide method(10.1 nM).Furthermore,we also validated the potential of this method for HDAC inhibitor screening and applied it to the analysis of HDAC activity in cell lysates.In conclusion,this study provided a simple and rapid new bioassy for the sensitive analysis of HDAC activity and the development of HDAC-targeted drugs.3.Design and construction of Cas12a molecular switch in response to protease activity based on Acr protein.AcrVA4 can bind to the Cas12a/crRNA complex and hinder the formation of the R-loop,thereby blocking the activation of the Cas12a’s function.In this chapter,based on the computational analysis and experimental verification,we fused AcrVA4,protease substrate sequence,and Cas12a to construct a Cas12a molecular switch that responds to protease activity.Tethering AcrV4 to Cas12a can effectively increase their local concentration s,leading to the efficient inhibition of Cas12a.When the target protein cleaves the linked polypeptide chain,the two proteins will be separated to restore the function of Cas12a.We further theoretically analyzed the amino acid residues on the interface between AcrVA4 and Cas12a and found that permutation of some of the amino acid residues of AcrVA4 could increase the activation efficiency.In addition,we further explored the scalability of this strategy and constructed a series of protease-responsive Cas12a molecular switches based on AcrVA5.Therefore,we developed a general Cas12a molecular switch construction strategy based on Acr protein,which provides a new paradigm for the development of responsive CRISPR-Cas systems.4.Cas12a molecular switch-mediated protease activity assay and responsive gene editing.We replaced the polypeptide linker to construct a panel of Cas12a molecular switches in response to different proteases,including hepatitis C virus(HCV)NS3/4A protease,rhinovirus(HRV)3C protease,tobacco etch virus(TEV)protease,thrombin(THB),matrix metalloproteinase 2(MMP2).Based on the signal amplification effect originating from Cas12a’s trans-cleavage activity,the above-mentioned Cas12a molecular switches can be used to construct assays for sensitive protease detection using fluorescent single-stranded DNA as the signal reporter.This method had a linear response to HCV protease in the concentration range of 0.02-5 nM with a detection limit of 1.95p M.Taking the anti-HCV drug Danoprevir as an example,we demonstrated the potential of this method in the screening of protease inhibitors.Subsequently,we also verified that the Cas12a molecular switch with responsiveness MMP2could differentiate the expression levels of MMP2 in normal cells and cancer cells,suggesting its application potential in the highly sensitive analysis of protease biomarkers.Finally,we also explored the controllable gene-editing mediated by Cas12a molecular switches in response to the target protease.After the simultaneous transfection of AcrVA4-Cas12a(TEV)and TEV protease plasmids,efficient gene editing could be detected in the cellular genome.In summary,we achieved a highly sensitive analysis of protease activity and protease-induced controllable gene editing based on the Cas12a molecular switch.