Development And Application Of Miniaturized Cas13 Protein Based On IDC Strategy

The CRISPR-Cas system has been developed to be a revolutionary gene-editing technology that has been developed for basic research,therapeutics,nucleic acid detection,diagnostics,imaging,and antiviral applications.The CRISPR/Cas13 system is an RNA mediated nuclease targeting single stranded RNA(ss RNA),which exhibits pre cr RNA processing activity and RNA targeting activity.The size of Cas13 protein is generally between 950 AA and 1300 AA,which is relatively large.However,the CRISPR-Cas editing systems for the therapeutic applications remain challenging due to the in vivo delivery constraints of adeno-associated virus(AAVs).Therefore,the development of compact Cas proteases is urgent.At present,several successful strategies for miniaturizing Cas proteins have been proposed in relevant studies.This mainly includes: 1)developing naturally compact Cas proteases in the metagenomic dataset.However,these compact proteins don’t completely recapitulate the advantages of previously optimized Cas proteins.2)the functionalities of Cas enzymes are modified by removing unnecessary functional domains.However,in this strategy,the Cas DNA-/RNA-cleavage activity is commonly eliminated,whereas its DNA-/RNA binding activity is preserved.3)By generating a random deletion mutant library to present the overall deletion of Sp Cas9,the mutants only retained about 72% and 63% of the Sp Cas9 protein sequence,respectively,with a corresponding decrease in DNA binding and cleavage activity.4)By constructing surface-localized deletion variants of poorly conserved Cas13 d orthologs allow protein size reduction with minimal loss of efficiency,this process is largely restricted by the available fragments being unable to meet the miniaturization requirements of a Cas protein for future applications.Due to the aforementioned challenges,it is necessary to develop a miniaturization strategy for gene therapy.Based on the above issues,this study aims to develop new miniaturization strategies to develop compact Cas proteins,and construct corresponding RNA editing systems and gene therapy using miniaturized Cas13 proteins.Firstly,we propose an IDC strategy for protein miniaturization based on protein structure and AlphaFold2,with the goal of maximizing protein miniaturization while preserving protein function.The IDC strategy is established by analyzing protein nucleic acid interactions,conformational dynamics,and protein family conservation in protein structure."I" represents "interaction".When Cas13 protein functions,it undergoes a process from a single protein to binding with cr RNA to form a binary complex,and then binding with target RNA to form a ternary complex.The entire functional process of Cas13 mainly revolves around RNA.Therefore,in terms of protein structure,the binding sites that interact with RNA and the scaffold structural units that support the binding sites are crucial."D" represents "Dynamic".During the functional exercise of Cas13 protein,various functional domains exhibit varying degrees of conformational activity.Thus,it is necessary to analyze the changes in various binding sites and scaffold structural units during the transformation process.By analyzing the binding sites and scaffold structural units in the dynamic process,we can exclude the entire protein structure and generate a compact Cas13 d protein."C" represents "Conservation".The Cas13 protein family has a high degree of structural conservation,so the analysis of "I" and "D" above can be applied to protein families with high structural conservation.So,the above three analysis processes "Interaction","Dynamic",and "Conservation" were refined,and a protein miniaturization strategy-IDC strategy was proposed.Secondly,based on the IDC strategy and AlphaFold2 prediction structure,three compact Cas13 d variants were successfully generated.Three mini Cas13 d variants exhibit complete RNA binding and cleavage activity,with consistent knockdown efficiency in 293 T cells.Subsequently,in order to expand the application scope of the IDC strategy,we generated mini Cas13 b variants targeting two widely used Cas13 b proteins.Two variants of mini Cas13 b retained enzyme activity comparable to wildtype proteases.In addition,we systematically investigated the in vivo and in vitro functional activity of mini RfxCas13 d.The results showed that the active function of mini RfxCas13 d was not only similar to that of RfxCas13 d,but also had high fidelity activity compared to RfxCas13 d,and its off-target rate was significantly reduced.Further utilizing the mini RfxCas13 d protein,an RNA base editor mini Vx was constructed,which has the same A-I editing ability as Vx and does not cause widespread base mutations in the transcriptome.Finally,by co packaging mini RfxCas13 d and cr RNA into AAV virus(adeno-associated virus),the expression level of Pcsk9 in mouse liver was successfully reduced.This indicates that the mini RfxCas13 d protein can successfully exert its activity in animals and can be used for gene therapy.In summary,this study successfully proposed the IDC strategy by analyzing AlphaFold2 to predict and analyze the structure of Cas13 protein,and used the IDC strategy to generate five compact variants with complete RNA binding and cleavage activity for Cas13 b and Cas13 d families.Meanwhile,it was found that mini RfxCas13 d is a high-fidelity active Cas protease that can be used for RNA editing and gene therapy.This study provides new strategies and tools for the development and application of compact proteins in future gene therapy.