Construction Of Cas13 RNA Editing System For Acute Lung Injury Therapy

Background and Objective:The traditional methods for the treatment of acute lung injury(ALI)include ventilator-assisted,extracorporeal membrane oxygenation,and so on.Although it can alleviate the progression of the disease,it lacks the initiative of treatment.After ALI,alveolar epithelial type 2 cells(AEC2s)proliferate and differentiate into alveolar epithelial type 1 cells(AEC1s)to form alveolar-like tissue,thus contributing to the restoration of damaged lung tissue.Therefore,guiding the regeneration of alveolar tissue is the key to the active treatment of lung injury.Regulating related transcription factors to enhance the repair ability of AEC2 s by gene editing may be a new strategy for the effective treatment of ALI.The CRISPR-Cas system from bacteria has been developed as an efficient gene-editing tool.In particular,the CRISPR-Cas13 system-mediated gene editing technology only targets editing the RNA of the gene to regulate cell function.Since the editing of targeted RNA will not cause permanent changes in genetic material,the use of gene editing tools derived from CRISPR-Cas13 system to regulate the function of key transcription factors in AEC2 s can provide new strategies for the restoration of lung injury.The purpose of this study is to establish an efficient and safe Cas13 X.1 editing system,select important transcription factors for targeted editing,analyze and evaluate Cas13 X.1editing for AEC2 s,and illuminate the application potential of this editing platform in the field of lung injury repair.We have established a Cas13 X.1-mediated A-to-I base editing system targeting CTNNB1(cadherin associated protein beta 1)transcripts,a C-to-U base editing system targeting FOXO3(Forkhead box O3)transcripts,and a Cas13 X.1 RNA knockdown system targeting CTNNB1,TKT(transketolase),and m Cherry.Based on the previous research and literature,FOXO3,the main downstream effector of PI3K/AKT signal pathway involved in lung tissue regeneration,and CTNNB1,the coding gene ofβ-catenin protein in Wnt signal pathway,were selected as target genes to explore the biological function of RNA editing techniques.So as to explore the feasibility of endogenous therapy based on AEC2 s proliferation and differentiation.The corresponding transcripts were edited by base editing system to change the amino acids at the phosphorylation sites of FOXO3 protein and β-catenin protein,prevent the phosphorylation and decomposition of FOXO3 and β-catenin protein,and make FOXO3 and β-catenin protein play a continuous role in mediating cell proliferation.Among them,the FOXO3-targeting base editing system did not achieve the expected efficiency,and the subsequent cell function verification was not carried out.The base editing for CTNNB1 achieved a high editing effect,and after editing,the cell Wnt/β-catenin signal activity was significantly increased,the intracellular β-catenin protein was increased,and the cell proliferation ability was enhanced,which has important value in the field of lung injury repair.Methods:The Bbs I site on the Cas13 X.1 skeleton plasmids was cleaved by Bbs I enzyme to form linear plasmids to be inserted with Spacer sequence.Based on the FOXO3,CTNNB1,m Cherry,and TKT gene sequence information queried in NCBI database,we designed Spacer sequences for FOXO3,CTNNB1,m Cherry,and TKT genes on the open-source g RNA online design platform.The Cas13 X.1 editing system was constructed by inserting the Spacer sequence into the Cas13 X skeleton plasmid through DNA ligase.The editing system was delivered into HEK293 T cells by liposome transfection,and the editing efficiency was evaluated by q PCR,Western Blot,next-generation sequencing,and digital PCR sequencing.CTNNB1 was transfected into human type 2 alveolar epithelial cells(A549)for the study of acute lung injury.We examined the proliferation of edited A549 cells using the CCK-8 kit.Then the mechanism of Cas13-mediated base editing promoting A549 cell proliferation was verified by immunofluorescence technology and bifiluminase reporter technology.Finally,we evaluated the safety of the editing system by analyzing the morphological changes of the edited cells,ribosomal RNA integrity detection,apoptosis analysis,and necrosis analysis.Results:1.Cas13 X editing platform including A-to-I base editing system,C-to-U base editing system and Cas13 X.1-based knockdown editing system was successfully constructed.Efficient editing of CTNNB1,TKT,and m Cherry transcripts was realized.Among them,the editing system Cas13 X.1 for CTNNB1 has achieved an editing efficiency of nearly 40%,which is a great improvement compared with the published editing system Cas13b(25%).In addition,the knockdown efficiency of Cas13 X.1 on the target transcript is up to 50%,which is equivalent to the traditional RNAi technology.Moreover,the Cas13 X system theoretically has a lower miss rate,which makes it more advantageous for future use in targeting ALI therapeutic sites.The editing efficiency of C-to-U single-base system targeting FOXO3 transcripts was low,which may be attributed to the fact that Cas13 X.1system still has certain selectivity for target genes.2.The CTNNB1 single base editing system was delivered to A549 cells by plasmid transfection.48 hours later,the proliferation of A549 cells was detected by CCK-8 kit.The results showed that the proliferation ability of A549 cells was significantly improved.Subsequently,the mechanism of Cas13 X.1 base editing system promoting cell proliferation was verified in tool cell HEK293 T.We performed immunofluorescence staining ofβ-catenin protein and detected the activation level of Wnt/β-catenin signal pathway in HEK293 T transfected with corresponding plasmid.The results showed that the Cas13 X.1editing system changed the phosphorylation site of β-catenin protein through CTNNB1(c.121 A > I),prevented the phosphorylation and degradation of β-catenin protein,led to the accumulation of β-catenin protein in the nucleus,and continuously activated Wnt/β-catenin signal pathway,thus promoting cell proliferation.Therefore,CTNNB1 was a vital target gene for Cas13 system in the restoration of acute lung injury.3.The safety of the Cas13 X.1 editing system is verified at the cellular level,which is the basis for the subsequent Cas13 X.1 system to be used in the repair of ALI in vivo.When the Cas13 X.1 system played an editing role in the cells,we evaluated the potential cytotoxicity of the system.From the evaluation of cell morphology,apoptosis,and cell necrosis after editing,it was confirmed that Cas13 X.1 system did not affect the normal morphology of cells and did not induce apoptosis or necrosis.In addition,ribosomal RNA integrity analysis of edited cells showed that Cas13 X.1 had little collateral activity,which is the main source of cytotoxicity.Conclusion:Cas13X.1 editing platform can not only be directly used in the restoration of acute lung injury,but also as a potent tool for efficient screening of potential therapeutic sites.CTNNB1 is an important target of Cas13 X.1 system for lung injury repair.It is feasible to induce the proliferation of AEC2 s through base editing system to mediate ALI endogenous therapy.There are still some obstacles in the application of Cas13 X.1 system to the repair of lung injury,such as poor editing efficiency of some genes,and it takes time for the editing system to express in vivo(it is difficult to cope with rapidly deteriorating acute lung injury).