CRISPR/Cas9-mediated Gene Editing In Generation Of FoxJ1-deficient Primary Murine Airway Epithelial Cells And Genetically Modified Ferret Models

Gene editing refers to gene manipulation techniques for base-pair deletion,insertion,site-directed mutagenesis and combinatorial editing of the nucleotide sequence of a target gene by the endogenous genomic repair mechanism in cells.The recently emerged CRISPR/Cas9 technology highly impoved the efficiency of gene editing.As an accurate targeted gene editing technology,it can not only promote the study of gene function,human disease models building,gene therapy,but also accelerate the genetic improvement of crops and animals.In traditional respiratory stem cell research,it is necessary to use a pure cell population with a same genotype as the research object.But,the purification of gene knockout cells is time-consuming,labor intensive,and has a high chance of failure.This has always been a major problem in applying gene editing technology to stem cell research.In this study,we combined the advantages of fluorescent reporter genes with the characteristics of CRISPR/Cas9-mediated gene knockout technology to develop an effective gene-knockout-cell enrichment platform.First,the sgRNA(LoxP-sgRNA)that efficiently targets the Loxp site was screened and transfected into ROSA26-LoxP-tdTomato-stop-LoxP-EGFP(ROSA-TG)reporter murine airway epithelial cells expressing Cas9.The results showed that CRISPR/Cas9 efficiently mediated the tdTomato gene cleavage in the middle of 2 Loxp sites,and about 72.7%of red fluorescent cells are transformed into green fluorescent or colorless cells.The sequence between the 2 Loxp sites in the genome of the transfected cell was amplified and subcloned,and it was confirmed by sequencing that most of the clones were double-digested(96.6%).Next,the above ROSA-TG cells were co-transfected with LoxP-sgRNA and 2 sgRNAs both targeting the exon 2 of mouse FoxJl gene.Green fluorescent transformed cells were selected by flow cytometry as FoxJl knockout enriched cells.The enrichment effect of the platform was evaluated by sequence analysis between the two targeting sites in FoxJl gene from all the three group of cells,which indicated that all the clones derived from green fluorescent cells were targeted for double site digestion in the FoxJl gene(26/26),while only 25%of red cells have double digestion(7/28).Gene expression analysis further confirmed that no transcriptional FoxJl mRNA was detected in green fluorescent cells.The results of epithelial cell differentiation assay showed that 4.32%of the green fluorescent cells differentiated into the ciliary cell,while the ciliated cells from red fluorescent cells accounted for 38.5%.Analysis of epithelial cell electrophysiological Chloride channel function mediated by cystic fibrosis transmembrane transport regulator(CFTR)further indicated that CFTR-mediated activated and inhibited short-current were 7.21 ?A/cm3 and 13.4 ?A/cm3 in green cells.While in the red cells,the activated and inhibited short-current were 33.6 ?A/cm3 and 64.5 ?A/cm3 which was 4.66 and 4.81 times of green cells respectively.This indicates that knocking out of the FoxJl gene has a significant inhibitory effect on the CFTR chloride channel after cell differentiation.Additionally,in order to expand the application of this gene knockout cell enrichment platform,we have also screened the sgRNA for knocking out the EGFP and tdTomato genes.The quenching of red and green fluorescence can be used as an alternative screening label.Mice are genetically more distant from humans in the physiology,cell component and cell biology in airway than ferrets.The smooth muscle actin expressing respiratory myoepithelial cells(aSMA+)have recently been identified as an important stem cell subset in respiratory epithelium,which play a major role in airway epithelial repairing after injury and maintaining airway homeostasis.Base on the previous work of establishing the CRISPR/Cas9 gene editing enrichment platform,we further constructed 2 reporter ferret models--ROSA-TG and aSMA-CreERT2 strains.These models will contribute to the study of myoepithelial stem cells development and the pathogenesis of chronic lung diseases.Technically,in order to explore a new method for constructing the ferret model quickly and efficiently by using CRISPR technology,this study therefore attempts to utilize two different DNA damage repairing mechanisms--Non-homologous end joining(NHEJ)and homologous direct repair(HDR)mediated knock-in when constructing these transgenic ferret models.In generating the ROSA-TG strain,three sgRNAs targeting the ROSA gene were designed in the ferret genome,and the optimal one was screened by in vitro digestion.The constructed donor plasmid was digested into single-stranded DNA and microinjected into 179 ferret fertilized eggs together with sgRNA and Cas9.A total of 151 fertilized eggs that developed to the 2-cell stage were transplanted into five pseudopregnant recipients,resulting in 23 newborns.Four healthy ROSA-TG transgenic hybrid ferrets were screened and confirmed by PCR genotyping,sequencing analysis and Southern blot.The viability and accuracy of the knock-in Cre/LoxP fluorescence reporter system was confirmed by fluorescent switch and Southern blotting analysis after infecting the ferret fibroblast cells with adenovirus expressing Cre recombinase.Similarly,in the generation of the aSMA-CreERT2 strain,the best sgRNA targeting the ACTA2 gene in ferret genome was designed,screened,and microinjected into 155 ferret fertilized eggs with the constructed single-stranded DNA donor plasmid and Cas9.110 fertilized eggs that developed to the 2-cell stage were transplanted into 5 pseudopregnant recipients,and a total of 16 ferrets were produced.Four healthy hybrid ferrets with transgene integrated were screened and confirmed by PCR genotyping,sequencing analysis and Southern blotting.Using a Cre reporter expressing lentivirus infection of aSMA-CreERT2 transgenic fibroblast,the expression of aSMA activated by adding TGF? protein and tamoxifen-assisted Cre nuclear transfer,it was confirmed that all the elements of genome integration are fully functional.CRISPR/Cas9 technology is famous of no species restriction,high specificity and efficiency.This study took advantages of CRISPR/Cas9 technology to conduct a sequencial pilot projects step by step.The content was established from gene knock-out murine airway primary cell line construction to transgenic ferret model generation.The research object gradually expanded from mouse airway epithelial basal stem cells in vitro to ferret zygote and tissue-specific myoepithelial stem cells in vivo.The research method extends from fluorescent gene knockout to using NHEJ and The HDR pathway to knock-in reporter gene.These studies lay a strong foundation for application of CRISPR and transgenic ferret model in the stem cell field.In the future,the lineage tracing study of myoepithelial stem cells will reveal the mechanism of respiratory myoepithelium development,function and hyperplasia.It will also provide a technical method and an effective model for the preparation of genetically modified primary cells,zygote and the study of airway stem cell biology using the ferrets model.