Study On The Characteristics Of PRPF8-RP Based On Gene Editing Technology As Well As Patient-derived IPSCs And One-stop RO

Objective:1.To generate induced pluripotent stem cells（i PSCs）and embryonic stem cells（ESCs）about retinitis pigmentosa（RP）carrying PRPF8（c.C5792T）point mutation by gene editing.To explore the repair of i PSCs from RP patients carrying PRPF8（c.C5792T）heterozygous mutations by different gene editing methods.2.To elucidate the effect of heterozygous mutation of PRPF8（c.C5792T）on retinal pigment epithelium（RPE）derived from i PSCs.To observe the characteristic restoration of i PSCs-RPE by correcting the PRPF8（c.C5792T）mutation site.3.To explore the effect of three-dimensional（3D）printed derived Polydimethylsiloxane（PDMS）microwell platform in the establishment of one-stop retinal organoids（ROs）.4.To investigate the feasibility of 3D printing PDMS mold in generating one-stop PRPF8（c.C5792T）ROs.Methods:1.We transfected CRISPR/Cas9 plasmids carrying sgRNAs and homology-directed repair（HDR）templates into RP patient-derived hiPSCs by electroporation.Subsequently,the clones that successfully repaired the mutation site were selected.Furthermore,we established i PS cell lines of the same mutation site（PRPF8（c.C5792T）heterozygous）from normal i PSCs using the same gene editing methods as above.2.We transfected adenine base editors（ABE）plasmids into RP patient-derived hiPSCs by electroporation without HDR templates.The clones that successfully repaired the mutant sites were selected through subsequent screening and cell lines were established.3.ESCs carrying the Cas9 reporter gene were pretreated with Doxycycline to activate Cas9 protein expression.And then homozygous PRPF8（c.C5792T）mutant ES cell lines were generated by electroporation with the sgRNA and homologous recombinant template only.4.i PSCs were differentiated into RPE cells（i PSCs-RPE）by induction differentiation.i PSCs-RPE morphology was observed,and cell transmembrane resistance and permeability were evaluated to reflect the barrier function of cells.RNA-Seq assay and verified test were performed to demonstrate the characteristics of cell transcriptome changes.5.The PDMS microwell platform suitable for ROs culture was constructed through 3D printing technology.And a one-stop long-term culture of ROs was conducted.The differences between the adherent 3D ROs generated by the PDMS microwell platform and the suspended 3D ROs generated by the agarose microwell platform were analyzed by RNA-Seq.The fetal bovine serum（FBS）was replaced by human platelet lysate（HPL）.The ROs carrying PRPF8（c.C5792T）heterozygous mutation were established for long-term culture.6.Values were obtained from three or more samples and were expressed as the mean±standard deviation（SD）.Statistical analyses were conducted using Graph Pad Prism 9.RT-q PCR data were analyzed using Light Cycler 96 software.And Western Blot results were quantified using Image J.ZEN 3.2（blue edition）software was used for analysis of ROs.Differences between two groups were carried out Student’s unpaired two-tailed t-test.P<0.05 was considered significant.Results:1.To explore the use of different gene editing methods to construct and repair i PS cell lines:（1）Using CRISPR/Cas9 gene editing technology to generate i PS cell lines from hiPSCs of normal origin,which were carried PRPF8（c.C5792T）heterozygous mutation;（2）A homozygous mutant ES cell line with the mutation site of PRPF8（c.C5792T）was established by using the Cas9 gene carrying ESCs;（3）Using CRISPR/Cas9 gene editing to repair PRPF8（c.C5792T）heterozygous mutations at patient-derived hiPSCs;（4）ABE gene editing was used to repair PRPF8（c.C5792T）heterozygous mutations in patient-derived hiPSCs.Finally,the off-target sites of the generated cell lines were analyzed and predicted.The results showed that the PRPF8-Cas9-corrected cell line established by CRISPR/Cas9 gene editing has a heterozygous mutation at the genomic chr1:26715393position.After the mutation site was corrected by gene editing,the expression levels of the PRPF8 gene and protein increased.2.Compared with the normal control group,the expression level of PRPF8 protein in i PSCs-derived RPE in patients with PRPF8（c.C5792T）heterozygous mutation was significantly decreased and reduced cell barrier function.Transcriptomic analysis revealed that the heterozygous mutation caused the up-regulation of P53-related genes and down-regulation of visual perception and ion transport-related genes.Na⁺/K⁺-ATPase fluorescence staining lost polarity,and intracellular Ca²⁺concentration decreased.However,abnormal disease phenotype of differentiated RPE cells recovered after ABE gene editing in i PSCs corrected the mutation site.3.PDMS microwell platform was established by 3D printing technology,which could realize one-stop differentiation of i PSC aggregates into adherent 3D ROs.Moreover,three regions of the neural retina（NR）,ciliary margin（CM）,and RPE could be produced within adherent 3D ROs without treatment of BMP4 and Matrigel.Retinal ganglion cells（expressing BRN3a）,amacrine cells（expressing AP2a）,horizontal cells（expressing PROX1 and AP2α）,photoreceptor cells for cone（expressing S-opsin and L/M-opsin）and rod（expressing Rod opsin）,bipolar cells（expressing VSX2 and PKCα）,and Müller glial cells（expressing GS and Sox9）gradually emerged in adherent 3D ROs.Finally,In workflow,we replaced FBS with HPL for ROs culture.4.The RO disease model with heterozygous mutation of PRPF8（c.C5792T）was established based on the PDMS microwell platform.Compared with the control group,the morphology of photoreceptor cells produced at the late stage of culture was abnormal.Conclusions:1.Using CRISPR/Cas9 gene editing and ABE gene editing techniques,heterozygous mutant i PSCs and homozygous ESCs of PRPF8（c.C5792T）were successfully established,and heterozygous mutant i PSCs of RP patient-derived PRPF8（c.C5792T）corrected.We found that ABE gene editing was more efficient than Cas9 gene editing,and CRISPR/Cas9 gene editing could be off-target.After the PRPF8（c.C5792T）mutation site was corrected by gene editing,the expression of the PRPF8 gene and protein increased.2.We found that the heterozygous mutation of PRPF8（c.C5792T）had damaging effects on the morphology and function of i PSCs-RPE,and the expression level of PRPF8 protein was significantly decreased in i PSCs-derived RPE from patients with PRPF8（c.C5792T）heterozygous mutation.Furthermore,ABE gene editing repaired the mutation site of patients at the i PSCs level,and the abnormal differentiated RPE cells were corrected,which could provide cellular resources for clinical translation.3.Based on the 3D printing of the PDMS mold,we successfully constructed one-stop uniform and homogeneous ROs for the first time.ROs with retinal structure could obtain by long-term culture.Adherent3D ROs containing NR-CM-RPE structure could produce without BMP4and Matrigel.Moreover,We established a xeno-free culture workflow for ROs culture by replacing FBS with HPL,which would generate ROs for clinical translation.4.A one-stop ROs disease model with heterozygous mutation of PRPF8（c.C5792T）constructed based on the PDMS microwell mold platform.This mutation resulted in morphological abnormalities of photoreceptor-associated cells in the late stage of ROs.