In Vivo Gene Editing Cas9/RecA Rescued Visual Function In Mouse Model For Retinitis Pigmentosa

Retinitis pigmentosa(RP),an inherited retinal disease characterized by the loss of rod and cone photoreceptors,is the leading cause of progressive vision loss and inherited blindness.Photoreceptors are sensory neurons within the retina that convert light signals into electrical signals for visual signal transduction.Patients with RP display signs of nyctalopia(night blindness)due to loss of rods,followed by loss of daytime vision due to secondary cone photoreceptor degeneration.The PDE6B gene,which encodes guanosine 3',5'-monophosphate(cGMP)phosphodiesterase 6b(PDE6B)for phototransduction,accounts for 4 to 5%of autosomal recessive RP.The rodless(rdl)mouse,which shows a point mutation(Y347X,C to A)in the Pde6b gene,is a widely used RP mouse model.The mutant mice exhibit early-onset and severe rod photoreceptor degeneration,leading to progressive visual loss and eventually total blindness.Thus,rdl mice are a suitable model for advancing therapeutic treatments for RP disease.Several strategies have been developed for rescuing photoreceptor degeneration in rdl mice,such as adeno-associated virus(AAV)vectors for gene complementation,or small molecules for promoting cell survival,even germline gene editing.However,each of these methods demonstrates substantial shortcomings such as limited efficiency or no gene correction for lasting phenotypic rescue.Moreover,most human genetic diseases are diagnosed at the postnatal stage.Therefore,alternative gene editing strategies with improved gene editing efficiency need to be established for precise gene editing in postnatal animal models.Clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 has been widely used in genetic engineering.The CRISPR-Cas9 technique relies on single-guide RNA(sgRNA)to direct Cas9 to generate DNA double-strand breaks(DSBs)at target loci,with the breaks repaired by either nonhomologous end joining(NHEJ)or the homology-directed repair(HDR)pathway,which requires a DNA template.NHEJ has been extensively used for gene deletion.However,NHEJ is an error-prone genome editing pathway that can introduce additional insertions and deletions(indel)in DSBs.Conversely,HDR is a high-fidelity pathway for genome editing of germline or proliferating cells,although it is considered inefficient and relies on cell division.However,most cells in postnatal tissues in mammals,including neurons,lose their proliferation ability along with maturation.Thus,the Cas9-mediated HDR pathway has rarely been applied in postnatal tissues and cells.Therefore,we aimed to design an HDR-based genome editing technique to precisely correct the gene mutation in postnatal rdl mouse retinae for RP disease therapy.The Escherichia coli recombinase A(RecA)protein can boost homologous recombination efficiency in mammalian and plant cells.RecA is an adenosine 5'-triphosphate(ATP)-dependent DNA binding protein that catalyzes DNA strand exchange reactions in homologous recombination.These facts prompted us to study whether the addition of bacterial RecA protein in Cas9-mediated genome editing could specifically increase the frequency of HDR.We developed a Cas9/RecA technique,which incorporated Streptococcus pyogenes Cas9(spCas9)and sgRNA-targeted RecA,to correct the gene mutation in postnatal rdl mice with enhanced HDR efficiency in vivo.Our research demonstrated that HDR efficiency was enhanced using the Cas9/RecA system both in vitro and in vivo.In addition,the system improved the HDR efficiency greatly in both mitotic and post-mitotic photoreceptor cells.It precisely corrected the DNA sequence and improved the survival of photoreceptor cells.Moreover,the analyses of pupillary light reflex(PLR)and ex-vivo electroretinogram(ERG)indicated that the visual ability of rdl mice was restored to a certain extent.In conclusion,this study established an improved gene repair system based on HDR,which can precisely repair the DNA sequence in vivo for post-mitotic neurons.It implies the feasibility to treat single-gene-mutation caused inherited retinal diseases by Cas9/RecA,which has important scientific and clinical significances.