Precise excision of expanded GGC repeats in NOTCH2NLC via CRISPR/Cas9 for treating neuronal intranuclear inclusion disease

Domain: biology
Published: 2026-01-13
Editorial outcome: minor_revision

Abstract
Neuronal intranuclear inclusion disease (NIID) is an adult-onset neurodegenerative disease caused by expanded GGC repeats in the 5’ untranslated region of the human-specific NOTCH2NLC gene. The high sequence similarity between NOTCH2NLC and its paralogs poses a significant challenge for precise gene editing. Here, we develop a CRISPR/spCas9-based gene-editing strategy that precisely excises the expanded GGC repeats in NOTCH2NLC without detectable off-target effects on the highly homologous NOTCH2/NOTCH2NL family genes (<2% sequence divergence at this locus). The efficacy, specificity and safety of this approach are rigorously validated across multiple experimental models, including human cell lines, NIID iPSCs, and our previously established transgenic NIID mouse model. Our results demonstrate that precise excision of the expanded GGC repeats effectively alleviates NIID-related neuropathological, molecular and behavioral abnormalities. This study establishes the proof of concept for genome editing as a therapeutic strategy for NIID and other related repeat expansion disorders.

Introduction
Repeatexpansiondisorders(REDs)representaclassofgeneticdiseases characterizedbythepathologicalexpansionoftandemrepeatsequen- cesbeyondtheirnormalrange1,2.Todate,morethansixtyREDshave beenidentifiedinhumans,withthemajoritymanifestingasneurode- velopmentalorneurodegenerativediseases,suchasfragileXsyndrome, Huntington’s disease, spinocerebellar ataxia and C9ORF72-related amyotrophiclateralsclerosisandfrontotemporaldementia1,3–8.Notably, recent advancements in long-read sequencing and whole-genome sequencing (WGS) technologies have significantly accelerated the dis- covery of new REDs9–12. Among these, GGC repeat expansions located withinthe“non-coding”regionsofthehumangenomehavegarnered particularattention13.One suchdisorder,neuronal intranuclear inclu- siondisease(NIID)hasattractedincreasingattentioninrecentyears.

NIIDisaprogressiveneurodegenerativedisordercharacterizedby widespreadeosinophilicintranuclearinclusionsinthenervoussystem andvisceralorgans14,15.Itexhibitssubstantialclinicalheterogeneity,with primarily neurological manifestations including dementia, muscle weakness,tremor,bradykinesia,cerebellarataxia,periodicencephalitic episodes,andautonomicdysfunction9,14–18,aswellasnon-neurological involvement19–21.Severecasescanleadtolossofambulationandpre- maturedeath14,17,18.In2019,theGGCrepeatexpansion(>60repeats)in the 5′untranslatedregion(5′UTR)oftheNOTCH2NLCgenewasreported asthegeneticcauseofNIIDbyseveralgroups9,22–24.Furthermore,this mutationhasalsobeenimplicatedinaseriesofneurodegenerativeand neuromusculardiseases22,25–32.Althoughcomprehensiveepidemiological dataarelimited,over600caseshavebeendocumentedworldwideby theendof202233.Despitethesegeneticinsights,noeffectivetreatments currently exist, and conventional pharmacological therapies provide onlylimitedsymptomaticreliefwithouthaltingdiseaseprogression.

PreviousstudieshaveconfirmedthattheGGCrepeatexpansions intheNOTCH2NLCgenearelocatedwithinanindependentupstream openreadingframe(uORF)positionedbeforethecanonicalATG,and these expanded GGC repeats can be translated into multiple toxic polypeptides,withpolyglycine(polyG)beingthepredominantone34. These toxic peptides accumulate as intranuclear inclusions in the centralnervoussystemandvisceralorgans,drivingdiseasepathology through mechanisms such as dysregulated alternative splicing, impaired nuclear-cytoplasmic transport, mitochondrial dysfunction, and abnormal ribosome biogenesis35–41. Consequently, targeting the expandedGGCrepeatsthroughgeneeditingrepresentsarationaland potentialtherapeuticstrategyforNIID.

The human-specific NOTCH2NL (NOTCH2 N-terminal like) gene family, which includes NOTCH2NLA, NOTCH2NLB, NOTCH2NLC and NOTCH2NLR, shareshighsequencehomologyandplayscriticalrolesin human brain development and homeostasis42–44. Specifically, NOTCH2NLA,NOTCH2NLB,andNOTCH2NLCareexpressedincortical progenitors, where they enhance self-renewal and delay premature differentiation by potentiating canonical Notch signaling, thereby contributing to the evolutionary expansion of the human cerebral cortex. Abnormal dosage of NOTCH2NL genes has been linked to neurodevelopmental disorders, including microcephaly or cortical malformationsuponlossandmacrocephalyorautismspectrumdis- ordersuponduplication.Therefore,itisessentialtospecificallytarget the GGC repeat expansion within NOTCH2NLC while preserving the normalexpression and functionoftheNOTCH2NLfamily. However, thenear-identicalsequenceflankingtheGGCrepeats(<2%divergence) among these homologous genes, combined with the large size and highGCcontentoftheexpandedrepeats,posesignificantchallenges forpreciseandefficientgeneediting.

In this study, we develop a CRISPR/spCas9-based gene-editing strategytospecificallytargettheGGCrepeatexpansionswithinthe NOTCH2NLCgene.Toensureeditingprecision,wedesignsgRNAswith minimalsequencehomologytoNOTCH2NLparalogsandNOTCH2and employed a dual-sgRNA approach. We systematically assess its effi- cacy,specificityandsafetyacrossmultiplemodels,includingHEK293 cells with normal GGC repeats or expanded GGC repeats in NOTCH2NLC, NIID patient-derived induced pluripotent stem cells (iPSCs) and neural progenitor cells (NPCs), as well as a previously established transgenic NIID mouse model. Importantly, targeted excisionoftheexpandedGGCrepeatsmarkedlyreducespolyGpro- teinlevelsandsignificantlyamelioratesneuropathological,molecular and behavioral phenotypes associated with NIID. Collectively, our workestablishesastrongpreclinicalfoundationforthedevelopment ofgenome-editing-basedtherapiesforNIID.

Results
CRISPR/Cas9-basedgene-editingspecificallytargetingGGC repeatswithinNOTCH2NLCinhumancelllineswithnormalGGC repeats

Given the near-identical sequence flanking the GGC repeats among NOTCH2NLC,NOTCH2 and other members ofthe NOTCH2NL family (NOTCH2NLA, NOTCH2NLB, and NOTCH2NLR, NOTCH2NLA/B/R for short), we conducted comparative analysis and identified two mis- matchregionsflankingtheGGCrepeats(SupplementaryFig.1A).To enablepreciseexcisionoftheexpandedGGCrepeatsofNOTCH2NLC, we designed multiple single guide RNAs (sgRNAs) targeting the upstreamanddownstreammismatchregions.Specifically,sgRNA1-4 weredesignedtotargetsequencesupstreamoftheGGCrepeats,while sgRNA 5-8targeted sequences downstream(Fig. 1A, Supplementary Fig.1B).ThedetailsofsgRNAsareprovideinSupplementaryData1.

First,thegene-editingefficiencyofeachsgRNAwasevaluatedin humancelllineswithnormalGGCrepeats(HEK293cells).Forty-eight hours after co-transfection with Cas9 and sgRNA plasmids (Supple- mentary Fig. 1C) at a Cas9-to-single sgRNA ratios of 4:1, which was selectedforpreliminaryscreenbasedonourpriorexperienceingenome editingofothergenes45.WeperformedaT7endonucleaseI(T7EI)assay on the PCR products amplified using NOTCH2NLC-specific primers (SupplementaryFig.1AandSupplementaryData2).Theresultsindicated that the upstream sgRNA1, as well as the downstream sgRNA5 and sgRNA6, exhibited optimal cutting efficiency (Fig. 1B–E). Intriguingly, unexpectedT7EIcleavagefragments(400–500bp)wereobservedinthe controlsampleswithoutCRISPR/Cas9targeting(Fig.1B,C).Sincethe extractedgDNApooledmultipleHEK293cells,theunexpectedbands mayreflectintrinsicvariationsintheGGCrepeatlengthofNOTCH2NLC amongcellswithinthesamepopulation(SupplementaryFig.2A).

TovalidatetheefficacyofthesesgRNAsmediatingGGCrepeats deletion, various sgRNA combinations were tested. T7EI assay per- formed on the PCR products amplified using NOTCH2NLC-specific primers demonstrated that both sgRNA1+5 and sgRNA1+6 combi- nationsefficientlyexcisedtheGGCrepeats(33–40%),asevidencedby truncated PCR products due to the GGC deletion (Fig. 1F–G) and mismatchcleavageproducts(SupplementaryFig.2B).Foreachcom- bination,wetesteddifferentCas9-to-sgRNAcombinationsratios(1:1, 2:1,and4:1)tooptimizethetargetingefficiency.The1:1ratiowasfound tobethemosteffective(SupplementaryFig.2C)andwassubsequently usedinallfollowingexperiments.

Then,weevaluatedwhethertheCas9-mediateddeletionofGGC repeats was specific to NOTCH2NLC in HEK293 cells. Genomic DNA region encompassing GGC repeats was amplified using consensus primersthatamplifyNOTCH2NLA/B/C/RandNOTCH2simultaneously (Supplementary Fig. 1A). The PCR products were subjected to TA cloning,andfiftyrandomlyselectedcoloniespersgRNAcombination weresubjectedtoSangersequencing.Theresultsconfirmedthatthe deletionofGGCrepeatsoccurredexclusivelyintheNOTCH2NLCgene, withnodetectableoff-targeteditinginhomologousNOTCH2NLA/B/R and NOTCH2 (Fig. 1H, I). The specificity of gene-editing wasfurther validatedbyPCRusingprimersthatspecificallyamplifyGGCrepeats region of NOTCH2NLA/B/R and NOTCH2. The agarose gel electro- phoresisconfirmedtheabsenceoftruncatedPCRproductsordele- tionsinthesehomologousgenes(Fig.1JandSupplementaryFig.2D). Inaddition,quantitativePCR(qPCR)analysisusingNOTCH2NLC-spe- cificprimers,orconsensusprimers,whichamplifyNOTCH2NLA/B/C/R andNOTCH2,demonstratedthatthedeletionofGGCrepeatsdidnot altertheexpressionofNOTCH2NLCoritshomologs(Supplementary Fig.3A),providingadditionalevidenceforthespecificityofthegene- editingstrategy.

Gene-editingofexpandedGGCrepeatssignificantlyreduced polyGlevelsinstableHEK293celllineexpressing NOTCH2NLC-98GGC

AccumulatingevidencesupportsthatthepolyGpolypeptides,trans- lated from the expanded GGC repeats, play a central role in the pathogenesisofNIID34–41.Toevaluatewhetherourgene-editingstrat- egy could effectively target the expanded GGC repeats and reduce polyG levels, we established a HEK293 cell line stably expressing NOTCH2NLC-98GGC-GFP(Fig.2A).TheGFPtag(startcodonremoved) was fused to the polyG for detecting its expression. PCR analysis confirmedtheexpansionofGGCrepeatsinthiscellline,andwestern blottinganalysisshowedthetranslationoftheexpandedGGCrepeats intopolyG-GFP(Fig.2B,C).Wethenco-transfectedthestablecellline withCas9andsgRNAcombinations(1+6,1+5)orcontrolsgRNAto testtheeffectofourgene-editingstrategy.

Westernblotanalysisdemonstratedasignificantreductioninthe levelsofbothaggregatedandsolubleformsofpolyG(by49.0–51.7% and 67.6–78.4%, respectively) following treatment with Cas9 and NOTCH2NLCsgRNAcombinations(Fig.2D,E).Consistentwiththese findings, immunofluorescence staining results showed that both NOTCH2NLCsgRNAscombinationssignificantlyreducedthenumber (by 63.1–69.7%) of polyG-GFP aggregates compared to the control group (Fig. 2F,G). Further PCR and sequencing analyses confirmed that the decrease in polyG-GFP signal was consistent with the suc- cessful deletion of GGC repeats (Supplementary Fig. 4A–E). Collec- tively,alltheseresultsindicatethatourgene-editingstrategytargeting expanded GGC repeats effectively and significantly reduced polyG expressioninvitro.

Gene-editingofexpandedGGCrepeatsreversedtranscriptomic changesinpatient-derivedcellmodel

Human iPSCs provide a more physiologically relevant system for studyingtheexpressionandfunctionofhuman-specificdiseasegenes thatareexpressedattheendogenouslevel.Wethereforeestablished iPSCs from peripheral blood mononuclear cells (PBMCs) of a NIID patient and a sex- and age-matched health control (Supplementary Fig.5A).Repeat-primedpolymerasechainreaction(RP-PCR)andGC- richPCR(GC-PCR)assay,performedaspreviouslydescribed22,deter- minedtheGGCrepeatlengthstobe113/17repeats(patient)and19/18 repeats(control)intherespectiveiPSClines(SupplementaryFig.5A, B).AlliPSClinesmaintainednormalkaryotypesandexhibitedcom- parable expression of pluripotency markers (Supplementary Fig.6A,B).

Recent studies have demonstrated a promising gene-therapy strategyinvolvingthecorrectionofexpandedCAGrepeatstonormalrepeatsviahomologydirectedrepair(HDR),whichhasbeensuccess- fully validated in a HD pig model46. However, whether this gene replacement strategy can be used in NIID models remains to be explored. To address this, Cas9 with sgRNA1+6 and a HDR donor templatewithnormalGGCrepeatswerenucleofectedintotheNIID iPSCline(SupplementaryFig.7A,B).Usingfluorescence-activatedcell sorting (FACS), serial dilution, and PCR-based genotyping, we suc- cessfullyisolatedfourmonoclonallinesinwhichtheexpandedGGC repeatswereeitherdeletedorrepairedtonormal-lengthrepeats.The deletedorrepairedGGCrepeatswerefurtherconfirmedbyRP-PCR, GC-PCR and Sanger sequencing after TA cloning (Supplementary Fig. 5C–F and Supplementary Fig. 7C–F). Furthermore, all GGC- deletion and GGC-repair iPSC lines, retained normal pluripotency andkaryotypicstability(SupplementaryFig.6A,B).

Toevaluatethespecificityofourgene-editingsystemandinves- tigatepotentialoff-targeteffects,weselectedtworepresentativeedi- ted iPSC lines, one with GGC-deletion (line-22) and one with GGC- repair(line-37),usingtheuneditedNIIDiPSClineasacontrol.First,we employeddeep-sequencing(deep-seq)toanalysistoptwentypoten- tialoff-targetsites(tenforsgRNA1andtenforsgRNA6),includingthe highlyhomologousNOTCH2andNOTCH2NLgenefamily(Fig.3A,B). ThesecandidatesiteswereamplifiedbyPCRusingcandidates-specific primers(Supplementarydata2)andsubjectedtodeep-seq.Bioinfor- matic analysis confirmed precise gene-editing in NOTCH2NLC, with minimaloff-targetactivity(Fig.3A,BandSupplementaryFig.8A,B). Second,tobroadlyassessgenomicintegrityandpotentialoff-target activity, we performed WGS on the same iPSC lines. Our results revealedcomparableandnegligibleextentofsingle-nucleotidepoly- morphism (SNP), insertion/deletion (InDel), structure variants and copynumbervariants(CNV)betweeneditediPSCsanduneditediPSC (SupplementaryFig.8C).Inaddition,read-depthanalysisoftheaddi- tional30predictedoff-targetsitesalsoshowednosignificantdiffer- encesbetweengroups(SupplementaryFig.8D,E),furthersupporting thespecificityofoureditingstrategy.

Subsequently, we differentiated these edited iPSCs (both GGC- deletion and GGC-repair) along with NIID and WT iPSCs into NPCs. ImmunofluorescenceanalysisshowedsimilarexpressionofPAX6and NestininGGC-deletionandGGC-repairNPCsrelativetoWTcontrols, suggestingpreservedneuraldifferentiationpotential(Supplementary Fig.9).Furthermore,nosignificantalterationswereobservedinthe expression levelsofNOTCH2NLA/B/C/Rand NOTCH2 following gene editing(SupplementaryFig.3B).

NIIDdevelopsneuronalpathologyinanage-dependentmanner. AlthoughourpreviousstudiesrevealedthatNPCsderivedfromNIID iPSCs retained differentiation capacity, they displayed significant transcriptomicalterationsthatmaybeassociatedwiththedisease39. ToinvestigatewhetherGGC-deletionorGGC-repaircouldreversethe transcriptomic alterations, we performed bulk RNA sequencing. Heatmap analysis and principal component analysis (PCA) revealed thatthetranscriptomicprofilesofGGC-deletionandGGC-repairNPCs morecloselyresembledthoseofWTNPCsthanNIIDNPCs(Fig.3C,D). Compared to WT NPCs, 1983 upregulated genes and 1244 down- regulatedgeneswereidentifiedinNIIDNPCs(Fig.3E).However,after GGC repeats deletion, 50.88% (633/1244) of downregulated genes increased and 74.74% (1482/1983) of upregulated genes decreased; whereas following GGC repeats repair, 72.59% (903/1244) of down- regulated genes increased and 70.30% (1394/1983) of upregulated genesdecreased(Fig.3F–I).GeneOntology(GO)analysisonthesubset genesthatexhibitreversaltrendsrevealedsignificantenrichmentin neuronalfunctionafterGGC-deletionandGGC-repair(Fig.3J).These findings suggest that removing or correcting the expanded GGC repeatsnotonlypreservesthepluripotencyanddifferentiationcapa- city of iPSCs but also reverses the GGC expansion-mediated tran- scriptomicchangesiniPSCsderivedcells.

Gene-editingofexpandedGGCrepeatsalleviateddisease- relatedpathologicalandclinicalphenotypesintransgenicNIID mousemodel

To evaluate the in vivo efficacy of our gene-editing strategy, we leveragedatransgenicNIIDmousemodel,previouslyestablishedin ourlaboratory,thatubiquitouslyexpressestheNOTCH2NLC-98GGC39. Thismodelfaithfullyrecapitulatespathologicalhallmarksandclinical features of NIID, including widespread intranuclear inclusions, pro- gressive neurodegeneration, motor deficits, as observed in patients withNIID39,40.TodelivertheCRISPR/Cas9systemeffectivelyacrossthe blood-brainbarrier,wepackagedCas9andsgRNAsintoAAV-PHP.eB,a capsidknownforitshightransductionefficiencyinthecentralnervous system47.

First,weconductedthetreatmentinaspecificbrainregionofNIID micetoevaluatetheinvivoeffectivenessofourgene-editingstrategy. To minimize inter-individual variability, therapeutic AAV (Cas9 and sgRNA1+6)andcontrolAAV(Cas9andcontrolsgRNA)wereinjected intotheleftandrightstriatumofthesameNIIDmouse,respectively. Injectionswereperformedatpostnatalday8–10(P8-10)withadoseof 1×1010 genomic copies (GC) per side. Four weeks post-injection, immunostaining revealed a significant reduction in the number of polyG aggregates on the left striatum injected with Cas9 and sgRNA1+6comparedtotherightsideinjectedwithCas9andcontrol sgRNA (Fig. 4A). Additionally, the striatum injected with Cas9 and controlsgRNAshowedsignificantdecreasedlevelsofGFAPandIBA1, markers of astrocyte and microglial activation, respectively, and a significant increasedNeuNexpression(Fig.4B–D).Consistentresults were observed when compared with age-matched non-disease con- trols,whichfurtherconfirmedthetherapeuticefficacyofourediting strategy(SupplementaryFig.10).

Subsequently, we conducted a systematic treatment via retro- orbital vein injection (ROV), a more clinically relevant injection method, in NIID mice. Previous studies have shown that NIID mice progressively develop phenotypic abnormalities starting around 40 days of age, with a median survival of ~2 months39. To assess the therapeutic potential of early intervention, we administered AAV (1×1011 GC per mouse) at P2 and performed a comprehensive eva- luation on treated NIID mice (injected with Cas9 and sgRNA1+6), untreated NIID mice (injected with Cas9 and control sgRNA) and control mice (injected with Cas9 and sgRNAs) at P35-50 (Fig. 5A). Immunofluorescence analysis confirmed successful transduction of AAVthroughoutthewholebrain(SupplementaryFig.11A).PCRper- formed with primers that amplify exon1 of NOTCH2NLC confirmed removalofexpandedGGCrepeatsinthegenomicDNAofmicepost ROVinjection(SupplementaryFig.11B).

We analyzed behavioral performance in mice at P35–P40 and foundthat,comparedtothecontrolmice,theuntreatedNIIDmice exhibitedsignificantlyreducedmovingdistanceanddurationinthe open-fieldtest(Fig.5B–E),aswellasmarkedlypoorerperformancein motor coordination tests such as the wire hang tests and rotarod (Fig. 5F–H). In contrast, the treated NIID mice showed significant improvement in these behavioral tests, with their performance reachinglevelscomparabletothoseofthecontrolgroup(Fig.5B–H). In addition, Kaplan-Meier survival analysis showed that the treated NIID mice exhibited significantly longer lifespans compared to the untreated NIID mice (Fig. 5I). Furthermore, western blot results demonstratedthat,comparedtotheuntreatedNIIDmice,thetreated NIIDmiceexhibitedsignificantlyreducedproteinlevelsofpolyGand GFAP,alongsideincreasedNeuNlevelsinthecortex,reachinglevels comparabletothoseofnon-diseasecontrolmice(Fig.5J,K).

Gene-editingofexpandedGGCrepeatsalleviateddisease- relatedtranscriptomicalterationsintransgenicNIID mousemodel

Transcriptionaldysregulationisawell-documentedmolecularhallmark ofNIID,whichisobservedinpatienttissues,patient-derivedcellsand animal models9,37–39. To investigate whether our CRISPR/Cas9-based strategytargetingexpandedGGCrepeatscouldreversetranscriptomic alterationsassociatedwithNIID,weperformedbulkRNA-seqanalysis oncortextissuesfromthetreatedNIIDmice(injectedwithCas9and sgRNA1+6), untreated NIID mice (injected with Cas9 and control sgRNA) and control mice (injected with Cas9 and sgRNAs). Global transcriptomic visualization via heatmap view and PCA analysis revealedthenotablyalteredgeneexpressioninuntreatedNIIDmiceas compared with the control mice, and these alterations could be reversedbyNIIDtreatmentwithCRISPR/Cas9(Fig.6A,B).Forexample, 1789upregulatedand334downregulatedgeneswereidentifiedinthe untreatedNIIDmicecomparedtothecontrolmice(Fig.6C).Incontrast, treatedNIIDmiceshowedmarkedlyfewerdifferentialexpressedgenes (DEGs,171upregulatedand134downregulated)(Fig.6D).Amongthe downregulatedgenes,52.10%(174/334)wereincreased,whileamong the upregulated genes, 58.69% (1050/1789) were decreased in the treatedNIIDmice(Fig.6E,F).Furtheranalysisrevealedthatthereversed DEGswereenrichedindisease-associatedpathways,includingneural functionandinflammatoryresponse,suggestingapartialrestoration followingGGCrepeat-deletiontreatment(Fig.6G–I).

Inaddition,weassessedtheimpactofourgeneeditingstrategy onperipheraltissue.PreviousstudiesindicatedthatpolyGaggregates arealsopresentincardiomyocytes,leadingtocardiacabnormalities40. Westernblotresultsshowedasignificantreduction(~79%)incardiac polyG protein levels in the treated NIID mice compared to the untreated NIID mice (Supplementary Fig. 12A). Consistent with our observationsinthecentralnervoussystem,transcriptomicprofiling demonstratedthatthetreatedNIIDmiceexhibitedgeneexpression patterns more similar to the control mice, as evidenced by both heatmapvisualizationandPCAanalysis(SupplementaryFig.12B,C). FurtheranalysisshowedthatthetreatedNIIDmicehadmarkedlyfewer DEGsthantheuntreatedNIIDmice(571vs5372)whencomparedtothe controlmice(SupplementaryFig.12D,E).Notably,56.53%(277/490)- 64.03%(3126/4882)oforiginallydysregulatedgenesdisplayedrever- saltrendsfollowingtreatment(SupplementaryFig.12F,G),withthese genesbeingsignificantlyenrichedinmitochondrialfunctionandcar- diaccontractionpathways(SupplementaryFig.12H).Takentogether, analysisofNIIDmicesuggeststhatdeletionofexpandedGGCrepeats effectivelyrescues transcriptomic dysregulation acrossboth central nervoussystemandperipheraltissue.

Methods
Humanparticipants
OneNIIDpatientandonehealthcontrolfrommainlandChinawere recruited in this study. Each participant underwent a neurological examination performed by at least two experienced neurologists. Thepatientwasa57-year-oldmalewithahistoryofbradykinesiaand hand tremors for over 10 years underwent physical examination, which revealed increased muscle tone in all four extremities and actiontremorsintheupperextremities.Cognitiveassessmentyiel- dedanMMSEscoreof28andaMoCAscoreof27.Thehealthcontrol was a 50 year-old male. Peripheral blood samples were collected fromallparticipantsafterobtainingwritteninformedconsent.These samplesweresubsequentlyreprogrammedintoiPSCsforresearch purposes.

GGCrepeatsizedetermination
TheGGCrepeatsizewithinNOTCH2NLCwasverifiedanddetermined using RP-PCR and GC-PCR, as previously described22. RP-PCR was performed with a FAM-labeled gene specific primer (CCTCAGCCC- GATACTCACCAT) and a repeat-containing primer [TACCAATACG CATCCCGCGATTTGTCTTA(CGG)5]; GC-PCR was performed with a FAM-labeled forward primer (AGCGCCAGGGCCTGAGCCTTTGAAG- CAG) and a reverse primer (TCGCCCCAGGTGGCAGCCCCGGGCGC CGCGGAC). Amplified products were analyzed by capillary electro- phoresis (3500xL Genetic Analyzer, Applied Biosystems) with allele sizingbasedontheGeneScan1000ROXSizeStandard.

Animals
ThetransgenicNIIDmousemodel(C57BL/6Jbackground)thatubi- quitouslyexpressesNOTCH2NLC-98GGCwas generated previously, andnosignificantdifferencesinpathologicalorbehavioralpheno- typesbetweenmalesandfemales39.HeterozygousNIIDmiceandthe littermatecontrolswereusedinthisstudy.Bothsexeswereusedfor stereotaxicinjection,ROV,andbehavioraltests.Allmicewerebred andmaintainedona12:12hlight/darkcycle(lightsoffat7p.m.)in the animal facility under specific pathogen-free conditions in the animal facility of Xiangya Hospital, Central South University. The temperature was maintained at 22±1°C with relative humid- ity(30–70%).

DerivationofiPSCanddifferentiationtoNPCs
PBMCs from NIID patient and healthy control were reprogrammed into iPSCs using CytoTune-iPS 2.0 Sendai virus reprogramming kit accordingtothemanufacturer’sprotocol.TheiPSCswereculturedon vitronectin-coatedplates in StemPro-34SFM(Gibco)medium.After 3–4weeks,themediumwasreplacedbyEssential8(Gibco)wheniPSC colonieswereestablished.TheiPSClineswerevalidatedbykaryotyp- ingtoconfirmchromosomalconstitution,andthespecificGGCrepeat expansionwithinthepatient-derivedlinewasverifiedbyRP-PCRand GC-PCR.IPSCsweredifferentiatedtowardtheneuralprogenitorline- ageaspreviouslyreported39.Inbrief,NPCswerederivedfromiPSCs using the monolayer culture protocol with STEMdiffTM Neural InductionMedium(Stemcelltechnologies).ThegeneratedNPCswere maintained in neural progenitor medium (STEMCELL Technologies) onMatrigel-coatedandusedforsubsequentexperiments.Allcelllines werenegativeformycoplasma.

Plasmidsandviruses
The NOTCH2NLC-98GGC, Cas9, and sgRNAs plasmids were con- structedinpreviousstudies39,45,46.ThesequencesofsgRNAarelistedin theSupplementaryData1.Thenon-targetingcontrolsgRNAwasver- ified previously45. Cas9 and sgRNA vectors were sent to PackGene Biotech(Guangzhou,China)forviralpackagingandproduction.Allthe viralvectorswerepackagedintothePHP.eBserotype.Thetitersofthe virusesusedforstereotacticinjectionandretro-orbitalinjectionwere 1×1013GC/mLand5×1013GC/mL,respectively.

Antibodies
Primary antibodies used in this study include the following: Flag (Sigma-Aldrich, F1804, 1:1000), vinculin (Sigma-Aldrich, V9131, 1:1000),NeuN(CellSignalingTechnology,24307s,1:1000),GFAP(Cell SignalingTechnology,3670S,1:1000),IBA1(Wako,019-19741,1:1000), GFP(Invitrogen,A-11122,1:2000),zsGreen(Sangon,D199984,1:2000), OCT4(Proteintech,60242-1-Ig,1:200),SOX2(Proteintech,11064-1-AP, 1:1000), PAX6 (Abcam, AB195045, 1:350), Nestin (CST, 33475S, 1:2000).Fluorescentsecondaryantibodies(1:200)usedweredonkey anti-rabbitanddonkeyanti-mouseantibodiesconjugatedwithAlexa Fluor 488 or 594 from Jackson ImmunoResearch. HRP-conjugated secondaryantibodies(1:10000)weredonkeyanti-rabbitanddonkey anti-mousefromJacksonImmunoResearch.

Cellcultureandtransfection
HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium supplementedwith10%fetalbovineserumandpenicillin/streptomy- cin(100u/ml)at37°Cwith5%CO .Forthetransfectionprocedure,the 2 cellswereseededonto12-wellplatesatadensityof~200,000cellsper well.After24h,thecellsweretransfectedwithplasmidsusingLipo- fectamine3000accordingtothemanufacturer’sinstructions.48–72h after the transfection, the cells were harvested for subsequent experiments.HEK293cells(Procell,CL-0001)wereauthenticatedby STR profiling and were tested negative for mycoplasma contamination.

Stablecelllineestablishment
200,000 HEK293 cells were co-transfected with a total of 1μg NOTCH2NLC-98GGC-GFP plasmid. DMEM was supplemented with G418(600μg/mL)forselection.48haftertransfection,thecellswere treated with trypsinand then reseeded in 96-well plates containing selectivemedium.Aserialdilutionapproachwasadoptedduringthis process.Theselectivemediumwasrefreshedevery3–4daystoelim- inate non-transfected cells while allowing resistant colonies to pro- liferate.OncevisibleG418-resistantfociemerged,theywereisolated and expanded sequentially into 24-well plates and later into 6-well platesforvalidationofstableintegration.

Nucleofectionandmonoclonalcellisolation
One day before the passage day, iPSCs were treated with 10μM Y-27632overnight.Onthepassageday,1×106iPSCsresuspendedin human stem cell nucleofector solution I (Lonza, Basel, Switzerland) were nucleofected with 2μg of plasmids (Cas9: sgRNA=1:1) using programB16inthenucleofectorIIbdevice(Lonza).Afternucleofec- tion,thecellsweretransferredtoafreshlyMatrigel-coatedplateand culturedinmTeSR1mediumcontaining10μMY-27632for1day,fol- lowed byregular mTeSR1medium.24–48hafter nucleofection,the iPSCssortedonaBectonDickinsonFACSAriaIIcellsorterwereseeded onto a 24-well plate layered with mouse embryonic fibroblasts at a densityof100cellsperwell.iPSCcoloniesemergedbetween10-and 12-days post-sorting. These colonies were transferred to freshly Matrigel-coatedplatesforgenotyping.Atotalofninetycloneswere analyzed and four monoclonal lines, in which the expanded GGC repeatswereeitherdeletedorrepairedtonormal-lengthrepeats,were selectedforsubsequentexperiments.

T7EIassay
T7 Endonuclease I assay was performed as described previously77. GenomicDNAwasextractedfromHEK293cellsusingacommercial DNAisolationkit(Magen).Toamplifythetargetregionsflankingthe CRISPR-Cas9editing site, PCR wasperformed with LA Taq (Takara) using primers specific to amplify NOTCH2NLC or NOTCH2/ NOTCH2NLA/B/R. The T7EI primers are listed in the Supplementary Data2.ThePCRproductswerepurifiedandsubjectedtoheteroduplex formation by denaturation and gradual reannealing to allow mis- matchedbasepairingbetweenthewild-typeandmutantalleles.The heteroduplexDNAwasdigestedwithT7EndonucleaseI(NewEngland Biolabs) at 37°C for 5min, followed by enzyme inactivation. The digestedfragmentswereseparatedona1.5%agarosegel,andtheband intensitieswerequantifiedusingtheImageJsoftwaretocalculatethe cuttingfrequency.

TAcloning
GenomicDNAregionencompassingGGCrepeatswasamplifiedusing primersthatamplifyNOTCH2NLA/B/C/RandNOTCH2simultaneously. The TA clone primers are listed in the Supplementary Data 2. PCR productswerepurifiedusingacommercialgelextractionkit(Magen). TAcloningwasperformedusingacommercialSuperTOPOTAclone kit (Vazyme).The ligation mixturewastransformed into chemically competentE.coliDH5αcells,followedbyplatingontoLBagarcon- tainingampicillinat37°Covernightforcolonyselection.Thenextday, colonieswerepickedandculturedinLBbroth.PlasmidDNAwasiso- lated and sequenced with M13 forward primer. Sanger sequencing resultswerealignedandanalyzedusingtheSnapGenesoftware.

ReversetranscriptasePCRandquantitativereal-timePCR
Total RNA was extracted from cell pellets using a TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was synthesizedfrom1μg oftotalRNAusingtheRevertAidFirst-Strand cDNASynthesisKit(ThermoFisherScientific)andoligodTprimers. Quantitativereal-timePCRwasperformedandanalyzedbyapplying the comparative cycle threshold method. The primers used for the quantitativereal-timePCRarelistedintheSupplementaryData2.

Westernblot
Mousetissuesorcellpelletswerelysedinice-coldRIPAbuffer(50mM Tris,pH8.0,150mMNaCl,1mMEDTApH8.0,1mMEGTApH8.0,0.1% SDS,0.5%DOC,and1%TritonX-100)containingproteaseinhibitor. ProteinconcentrationwasdeterminedwiththeBCAProteinAssayKit (Thermo Scientific). Protein samples were separated by SDS- polyacrylamide gel electrophoresis and transferred to a poly- vinylidenefluoridemembrane(Millipore),whichwasfurtherblocked with5%milk/PBSfor1hatroomtemperature.Theblotwasincubated withprimaryantibodiesovernightat4°C.Afterthreewashesin0.05% PBST, the blot was incubated with HRP-conjugated secondary anti- bodiesfor1hatroomtemperature.Afterthreewashesin0.05%PBST, protein bands were developed using an ECL Prime Chemilumines- cencekit(GEHealthcare)onaBio-Raddevelopingandimagingplat- form. Protein band intensities were quantified using ImageJ to calculateexpressionlevelsrelativetointernalcontrols.

Immunofluorescentstaining
Mice were anesthetized and perfused intracardially with 0.9% saline solution,followedby4%paraformaldehydein0.1Mphosphatebufferat pH7.2.Theisolatedmousebrainsweredehydratedin30%sucroseat 4°Candsectionedatathicknessof30μmforthesubsequentimmu- nofluorescencestudy.Brainsectionswereblockedwithasolutionof3% bovineserumalbumin(BSA)in0.3%TritonX-100/PBSfor60min,fol- lowed by incubation with primaryantibodies overnight at4°C. After incubationwithfluorescentsecondaryantibodiesand4′,6-diamidino-2- phenylindole(DAPI),thebrainsectionsweremountedontocoatedglass slidesandexaminedusingaZeissmicroscope(Apotome3).

ForimmunostainingofiPSCsandNPCs,cellsgrownoncoverslips werefixedwith4%paraformaldehydefor10minandpermeabilized with0.1%TritonX-100for15minatroomtemperature.Throughout theentirestainingprocess,asolutionconsistingof2%normalgoat serumand3%BSAinPBSwasusedforblocking.Incubationwithpri- mary antibodies was carried out overnight at 4°C and followed by fluorescentsecondaryantibody incubation for 45min at room tem- perature.ImageswerecapturedusingaZeissmicroscope(Apotome 3). The number of polyG aggregates and immunofluorescent inten- sitiesofGFAP,IBA1,andNeuNwerequantifiedusingImageJsoftware.

Stereotaxicinjectionandretro-orbitalveininjection
Duringthestriatalstereotaxicinjection,P8-10micewereanesthetized viainhalationof1.5%isofluraneandthenfirmlypositionedinastereo- taxicinstrument.AAV-sgRNAandAAV-Cas9weremixedataratioof1:1. 1μlofthemixedviruses(1×1010GC)wasinjectedintoonesideofthe mousestriatumaccordingtothefollowingcoordinatesadjustedtothe flatskullposition:0.39mmrostraltothebregma,1.4mmlateraltothe midline,and 2.5mmventralfrom the dural surface overa period of 5min.AnRWDstereotaxicinstrument(69100)andaHamiltonmicro- syringe(1700series,10μl)wereusedtodeliverthevirusataspeedof 100nlpermin.Afterthesurgery,themicewereplacedonaheated blankettorecover.Allstereotaxicsurgerieswerecompletedsuccess- fully,withincisionshealingwellandnoadverseeventsobserved.

ROVwasconductedfollowingapreviouslyestablishedprotocol78. Briefly,P1-P2neonatalmicewerecryo-anesthetizedonicefor3min. ThevirusesexpressingsgRNAandCas9weremixedataratioof1:1and 2μlofthemixedviruses(1×1011GC)wereinjectedintoeachpup.To performtheinjection,placethepupinleftlateralrecumbencywithits headfacingtotheright.Gentlyholdthepup’sheadwiththethumb and forefinger of the non-dominant hand. Identify the retro-orbital sinusregionandinsert~1/3ofthelengthofa10μLneedle,withthe bevel facing down, at a 45° angle into the inner canthus. After the injection,thepupswereplacedonaheatedblankettorecover.Among sixty-fourinjectedpups,fewerthantendiedduetopoorrewarming afterhypothermicanesthesiaorcannibalizationbythedam,whilethe remaining mice survived and underwent subsequent experiments. Injectionswereperformedinablindedmanner,asthegenotypeswere unknownatP1-2.Foreachlitter,eithertherapeuticorcontrolAAVwas administered uniformly. After genotyping, transgenic mice were assignedtoeithertreated(withtherapeuticAAV)oruntreated(with controlAAV)NIIDgroups,andallcontrollittermateswereassignedto thecontrolgroup.

Mousebehavioralanalysis
Behavioraltestswereperformedaspreviouslydescribed39.Atleast13 micepergenotypewereincluded.Theopenfieldtestwasperformedin aPerspexbox(40cminlength,40cminwidth,and31cminheight) withthefloordividedinto16equal-sizedsquares.Duringthe10-min testperiod, the movement of the mice was recorded on video and analyzedbyanautomatedbehavioranalysissystem.Thetotalmoving durationanddistancewerethencomparedbetweendifferentgroups.

Inthewire-hangtest,micewereplacedonthewirelidofamousecage, whichwastheninverted.Duringthe3-mintestperiod,thelatencyto fall was recorded. Each mouse underwent the test twice, and the average hang time was compared across different groups. For the rotarodtest,miceweretrainedfor3minontherotarod,withthree trials per day for three consecutive days. On the testing day, each mouseunderwentanaccelerating-speedtest(from5to20rpm)for 3min, and this test was repeated twice. The intertrial interval was 10min,andthelatencytofallwasrecordedforeachtrial.Theaverage timethemicespentontherotarodwascomparedamongdifferent groups. The survival time of each mouse was monitored and recordeddaily.

Deep-sequencinganalysis
Potentialoff-targetsiteswerepredictedusingtheonlinetoolCRISPOR. WegeneratedaPCRlibraryfornext-generationsequencing(NGS).We first used specific primers to amplify genomic DNA containing the targetingregionwithLATaq(Takara).ThesecondPCRwasthenper- formedwiththesameprimerscontainingIlluminaforwardandreverse adapters to generate the PCR library for NGS. The final 50μl PCR productswerepurifiedusingacommercialgelextractionkit(Magen) andqualifiedwithanAgilentBioanalyzer2100.ThePCRproductswere thensenttoIGEBiotechnology(Guangzhou)forsequencing.TheNGS datawereanalyzedbyCRISPResso279.Thetop20potentialoff-target sitesandprimersequencesarelistedintheSupplementaryData2.

RNA-seqandanalysis
Micecortexandheart,andhumanNPCswereusedforRNAextraction. Anamountof1μgoftotalRNApersamplewasprocessedforlibrary preparationusingVAHTSUniversalV10RNA-seqLibraryPrepKitfol- lowing the manufacturer’s recommendations. Libraries were sequencedonanIlluminaNovaSeqXPlus(micetissues)orIllumina Novaseq6000(humanNPCs)platform.Rawreadswereprocessedby fastp to get clean reads. Paired-end clean reads were mapped to GRCm38orGRCh38byHISAT2andassembledbyStringTie.Quanti- ficationofgeneexpressionlevelswasestimatedbyTPM(micetissues) orFPKM(humanNPCs).HeatmapandPCAanalysiswereperformed using R80. Differential expression analysis was performed using DESeq2.GOenrichmentanalysisofDEGswasperformedusingR81.

Whole-genomesequencing
WGSwasperformedbyNovogene,Co.,LTD.Atotalamountof0.2μg DNApersamplewasusedasinputmaterialfortheDNAlibrarypre- parations.SequencinglibrarywasgeneratedusingNEBNext®Ultra™ DNALibraryPrepKitforIllumina(NEB,USA)followingmanufacturer’s recommendationsandindexcodeswereaddedtoeachsample.Next, the DNA libraries were sequenced on Illumina platform and 150bp paired-endreadsweregenerated.Theeffectivesequencingdatawas aligned with the reference sequence through BWA82 software. Samtools83andbcftoolswereusedtoextractallpotentialpolymorphic SNPsandInDelsitesfromtheentiregenomewiththemappedreads. BreakDancer84softwarewasusedtodetectinsertion,deletion,inver- sion,intra-chromosomaltranslocationandinter-chromosomaltrans- locationmutations,basedonthereferencegenomemappingresults andthedetectedinsertsize.CNVnator85wasusedtodetectCNVsof potentialdeletionsandduplications.Cas-OFFinder86wasusedtopre- dictoff-targetsacrosstheentiregenome.Additional30off-targetloci intheexperimentalgroupwerecomparedwiththecontrolgroup.The relativedepthswerecalculatedbytakingthenumberofmappedreads onthesesitesintothewholegenomeofthepointerreads.

Statisticalanalysisandreproducibility
Allstatisticalanalyseswereperformedwitheithertwo-tailedStudent’s t test or the one-way ANOVA test with multiple comparisons using GraphPadPrism9.Allquantitativedatawerepresentedasmean±SEM. A P value of less than 0.05 was considered statistically significant. Samplesizeswerechosenbasedonestablishedstandardsinthefield to ensure robustness. No statistical method was used for pre- determination,andnodatawereexcluded.Forinvivostudies(e.g., behavior,survival;N≥8pergroup),littermateswererandomlyallo- catedandinvestigatorswereblindedduringproceduresandassess- ment.Forinvitroassays(N≥3independentreplicates),whilegroup allocationwasdefinedbyexperimentalconditions,sampleprocessing andanalysisorderwererandomized.Quantitativeanalysisforinvitro datawasperformedwithblindingtogroupidentity.

Ethicsstatement
Allexperimentalprotocolsinvolvinghumanparticipantsandanimals were conducted in compliance with all relevant ethical regulations. Thestudyinvolvinghumanparticipantswasreviewedandapproved bytheEthicsCommitteeofXiangyaHospital,CentralSouthUniversity (ApprovalNo.2024040420).Writteninformedconsentwasobtained from all human participants. The animal study was reviewed and approvedbytheAnimalEthicsCommitteeofXiangyaHospital,Central SouthUniversity(ApprovalNo.202103138).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThis study targets repeat expansion disorders, with a particular focus on neuronal intranuclear inclusion disease (NIID), a\nrecently recognized neurodegenerative disorder. The authors applied Cas9/sgRNA-mediated gene editing to specifically\ntarget GGC repeat expansions located in the uORF of NOTCH2NLC, demonstrating therapeutic potential across multiple\nmodels by directly addressing the root cause of the disease. The authors rigorously validate their approach across multiple\nindependent platforms, including engineered HEK293 cells, NIID patient-derived induced pluripotent stem cells (iPSCs),\nand a NIID transgenic mouse model, while incorporating comprehensive molecular, cellular, histological, behavioral, and\ntranscriptomic analyses. Notably, they demonstrate excellent gRNA specificity and off-target control—a critical consideration\ngiven the high sequence similarity (>98%) between NOTCH2NLC and other NOTCH2NL family members—supported by\nrobust experimental design and validation techniques such as sequencing and qPCR. In HEK293 cells, they validated the\nediting efficiency of each sgRNA, confirming that base editing reduces polyG expression, a pathological hallmark of NIID.\nNext, gene editing and replacement were performed in iPSCs derived from NIID patients, where transcriptomic profiling via\nvolcano plots demonstrated that on-target editing had minimal off-target effects, confirming high specificity. Subsequently, in\nvivo editing was carried out in a NIID mouse model, with retro-orbital injection at postnatal day 2 followed by behavioral\nassessments. Edited mice exhibited a reduction in polyG aggregates, alongside decreased GFAP expression and increased\nNeuN levels, mirroring observations from human cells. Finally, transcriptome-wide changes were analyzed in the mouse\nmodel, demonstrating that gene editing rescues transcriptomic dysregulation across both central nervous system and\nperipheral tissues. Importantly, the study provides compelling evidence for the functional relevance of their intervention by\nshowing clear reversal of disease phenotypes at both the molecular and behavioral levels, with the demonstration of\nlifespan extension in treated animals marking a major translational milestone. This work not only establishes a foundation\nfor NIID therapy but also holds significant promise for treating other GGC repeat expansion diseases. Although the study is\nsignificant and demonstrates important findings, there are comments that authors need to address as listed below.\nAbstract\n1. Lines 36-38, \"Here, we developed a CRISPR/spCas9-based gene-editing strategy to precisely excise the expanded GGC\nrepeats in NOTCH2NL...\" To be specific, it should be NOTCH2NLC, not NOTCH2NL.\nIntroduction\n1. Lines 89–97, the description of the function of the NOTCH2NL family genes can be developed more here. Specifically, it\nis informative to clarify why preserving NOTCH2NL family genes is important—e.g., which one(s) are involved in cortical\ndevelopment and must remain unaltered to avoid developmental deficits.”\nResults and Figures\n1. Line 123, one of the downstream targeted sequences do not match between the main text (864), Fig.1C (864), Fig.S1B\n(866) and Table.S1 (866). In addition, please describe what the numbers in the names of gRNAs mean. If their names are\nfrom their sequence, does the number mean the beginning of the sequences or the end of the sequences or something else.\nPlease also confirm if their names match with their actual number in their sequences. For example, it is puzzling what 702\nand 703 are named after since they are not 1 nucleotide apart, which could mis-lead authors intention to readers. Once\nauthors clarify/confirm the nomenclatures, the labels in the cartoon of Figure S1B, main text and the Figure Legends all need\nto be rechecked and revised accordingly.\n2. Lines 126-130, “First, the gene-editing efficiency of each sgRNA was evaluated in human cell lines with normal GGC\nrepeats (HEK293 cells). Forty-eight hours after co-transfection with Cas9 and sgRNA plasmids (Fig.S1C) at a Cas9-to-\nsgRNA ratio of 4:1…” what is the rationale behind choosing 4:1? If there is a reference that authors followed, please cite the\npaper. Otherwise, authors need to briefly clarify why 4:1 was chosen to begin with. Later (Line 145), it is mentioned that “the\nmost effective ratio was found to be 1:1”. Any reason that authors chose to test 1:1, 2:1, 4:1 instead of the opposite ratios for\nCas9 to sgRNA (e.g., more sgRNA to Cas9 ratio such as 1:2 or 1:4)? If authors found that 1:1 ratio is most effective among\ntested ratios, one could think that 1:2 could be even more effective if tested. Please clarify it.\n3. Human phenotypes: PolyG aggregation was assessed in both HEK293T cells and mouse tissues, yet it was not\nevaluated in patient-derived human cells. Given its pathological relevance and the consistency of analysis across models,\nthere appears to be no justification for omitting polyG detection in human fibroblasts. Including this data would strengthen the\ntranslational relevance of the findings and enable a more direct comparison across all experimental systems.\n4. Figure 2: Are the GFP level alterations demonstrated in this Figure solely due to the GGC shortening or could that be also\npartially due to the frameshift location after the shortening and leading GFP not be able to be expressed? Please clarify this\nin the main text. In addition, can the labels of poly G be modified to ‘GFP: PolyG’ instead of ‘PolyG’ as authors did in their\nprevious publication (Sci Adv, 2022) to avoid any misleading/overstatement?\n5. Figure 3: The numerical values presented in the text appear consistent overall; however, in Figure 3H and 3I, the sum of\nindividual dots within certain conditions does not exactly match the corresponding group totals. For example, in Figure 3I,\nthe sum of two data points in the “NIID down” group, it is 341 + 903 = 1244, and in the “GGC-rep down” group, it is 323 +\n1394 = 1717, which aligns well with the indicated totals. However, in other samples or in Figure 3H, the individual data\npoints do not sum precisely to the indicated total. Could the authors clarify whether the plotted values represent raw counts,\nnormalized data, or are derived from averages across replicates? Please revise accordingly and as needed.\n6. Figure 3: Can authors indicate what color means what in their Figure legends and can authors consider to use consistent\ncolor code for Figure 3I and 3H? For example, you can match the color of NIID down and NIID up between Figure 3I and 3H.\n7. Figure 4 and Figure S8: Authors should include age-matched wild-type control (or non-disease control group) to\ndemonstrate the phenotype of NIID mice as their control group (as authors did in Figure 5B-5I). Without comparing with WT\nmice, authors cannot conclude what authors have demonstrated in treated NIID mice as they rescued the neuropathological\nphenotypes. Additionally, what are the differences between Figure 4 and Figure S8? Can authors be clear about their\ndescription on Figure S8 if there is any difference from Figure 4? Even if authors wanted to demonstrate Figure S8 as\nanother representative Figure to support Figure 4, it is unclear if the Figure S8 was representative images as a part of the\nquantification result presented in Figure 4 or if Figure S8 was an independent experiment. If Figure S8 was an independent\nexperiment, please have their quantified result as a graph to support author’s conclusion about Figure S8. If not, please\nclarify in the main text and in the Figure legends.\n8. Figure 5J: The correlation between zsGreen expression, polyG reduction, and GFAP/NeuN changes appears\ninconsistent, making it difficult to link editing efficiency with phenotypic rescue. For example, second sample of Treated NIID\nmice has higher expression levels than other two mice, but their GFAP or NeuN levels looks fairly similar to each other\nbetween all 3 mice compared to how different they are from the second mouse. Authors need to clarify how or whether they\nnormalized their data to the levels of zsGreen. Comparing samples between mice which demonstrate comparable zsGreen\nexpression levels would improve data interpretation and strengthen conclusions.\n9. Figure 5K lacks a non-disease control group, making it difficult to evaluate the full extent of pathological elevation or\nrescue. The authors should include age-matched wild-type control group to establish a baseline and demonstrate whether\ntreatment restores polyG to the physiological levels.\n10. Figure S1A: The second ‘Upstream mismatch sequences’ should be revised to be ‘Downstream mismatch sequences’.\n11. Figure S3B: The X-axis labels indicate Untreated vs Treated. However, the description is missing in the Figure and\nFigure Legends about what they were treated with. More detail information about Figure S3B needs to be added to the\nFigure Legends. In addition, the X-axis labels need to be revised to make it clearer and consistent to other labels.\n12. Figure S4: The order in the main text and the Figure is RP-PCR then GC-PCR, which doesn’t match with the Figure\nLegend (GC-PCR then RP-PCR). Please revise the Figure Legend accordingly to match with other part of the manuscript.\n13. Figure S10A: Y-axis label is missing from the graph.\nMethods\n1. For animal study, were both sexes used, or only male or only female? If so, can authors describe it clearly in the method\nsection and justify the reason why they chose those genders.\n2. Antibody information: It is better to indicate dilution ratios for primary and secondary antibodies to increase reproducibility\nof the study.\n3. Were any mortality or adverse events occurred during stereotaxic surgery or during the behavior with animals who went\nthrough surgeries? If so, please describe it in the manuscript.\n4. Information of patient age/sex and number of clones analyzed would strengthen rigor. Please describe them as much as\nauthors can.", "reviewer_2": "In this work, Xie et al. have used a CRISPR/Cas9-based gene-editing approach to remove the expanded GGC repeats in\nthe NOTCH2NLC gene, which is the known genetic cause of neuronal intranuclear inclusion disease (NIID). To prevent off-\ntarget effects in homologous genes, the authors created and verified dual sgRNAs that flank the repeat region. HEK293\ncells, NIID patient-derived iPSCs, and a transgenic mouse model of NIID were used to validate the editing technique. The\nfindings demonstrated that the expansion of the repeat was efficiently removed, that pathological polyG aggregates were\nreduced, that transcriptomic profiles were restored, and that the neurobehavioral phenotypes in mice were reversed. The\nwork has great clinical translation potential and is backed by lots of phenotypic and molecular data.\nThe manuscript is strong in several ways. It presents a very specific, cleverly designed gene-editing technique and has been\nproven to work in a variety of biological systems. The findings' robustness and translational potential are enhanced by the\nuse of in vitro, ex vivo, and in vivo models. The paper offers a comprehensive examination that includes behavioral\nevaluations in animal models, transcriptome rescue, and off-target consequences. Furthermore, the work addresses\nimportant translational challenges like immune response and vector administration within a therapeutically relevant\nframework.\nOverall the study is well planned and carried out. The main conclusions are sound, novel, and I think that this work is of high\ninterest to the fields of genome editing and neurogenetics. I only have a few suggestions/remarks. First, only the top 14\nprojected loci are included in the off-target prediction. This could be improved by more thorough genome-wide off-target\nstudies, such GUIDE-seq or CIRCLE-seq. Maybe the authors could discuss this. Second, although the mouse model\nexhibits important characteristics, its limitations in replicating the human disease should be discussed in more detail. Third, it\nwould be good to include a summary table for sgRNAs, editing efficiencies, and off-target profiles.\nIn summary, my recommendation is accept with minor revisions.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We sincerely thank the reviewers for their highly positive comments, their recognition\nof the significance and insightfulness of our work. Their constructive critiques have\nbeen invaluable in refining our manuscript. We have thoroughly addressed all concerns\nraised, revising the text point-by-point and incorporating corresponding revisions\nthroughout.\nIn addition, we have integrated the new perspectives suggested by the reviewers and\nrefined the Discussion section to enhance its clarity and to more effectively convey the\nsignificance of our findings. We have highlighted all changes in blue in the revised\nmanuscript, and believe these revisions have significantly improved the overall quality\nof the manuscript. We hope the reviewers and editor now consider it suitable for\npublication in Nature Communications.\nThe detailed responses are shown below (in blue font).\nReviewer comments\nReviewer #1 (Remarks to the Author):\nSummary\nThis study targets repeat expansion disorders, with a particular focus on neuronal\nintranuclear inclusion disease (NIID), a recently recognized neurodegenerative disorder.\nThe authors applied Cas9/sgRNA-mediated gene editing to specifically target GGC\nrepeat expansions located in the uORF of NOTCH2NLC, demonstrating therapeutic\npotential across multiple models by directly addressing the root cause of the disease.\nThe authors rigorously validate their approach across multiple independent platforms,\nincluding engineered HEK293 cells, NIID patient-derived induced pluripotent stem\ncells (iPSCs), and a NIID transgenic mouse model, while incorporating comprehensive\nmolecular, cellular, histological, behavioral, and transcriptomic analyses. Notably, they\ndemonstrate excellent gRNA specificity and off-target control—a critical consideration\ngiven the high sequence similarity (>98%) between NOTCH2NLC and other\nNOTCH2NL family members—supported by robust experimental design and\nvalidation techniques such as sequencing and qPCR. In HEK293 cells, they validated\nthe editing efficiency of each sgRNA, confirming that base editing reduces polyG\nexpression, a pathological hallmark of NIID. Next, gene editing and replacement were\nperformed in iPSCs derived from NIID patients, where transcriptomic profiling via\nvolcano plots demonstrated that on-target editing had minimal off-target effects,\nconfirming high specificity. Subsequently, in vivo editing was carried out in a NIID\nmouse model, with retro-orbital injection at postnatal day 2 followed by behavioral\nassessments. Edited mice exhibited a reduction in polyG aggregates, alongside\ndecreased GFAP expression and increased NeuN levels, mirroring observations from\nhuman cells. Finally, transcriptome-wide changes were analyzed in the mouse model,\ndemonstrating that gene editing rescues transcriptomic dysregulation across both\ncentral nervous system and peripheral tissues. Importantly, the study provides\ncompelling evidence for the functional relevance of their intervention by showing clear\nreversal of disease phenotypes at both the molecular and behavioral levels, with the\ndemonstration of lifespan extension in treated animals marking a major translational\nmilestone. This work not only establishes a foundation for NIID therapy but also holds\nsignificant promise for treating other GGC repeat expansion diseases. Although the\nstudy is significant and demonstrates important findings, there are comments that\nauthors need to address as listed below.\nAuthor response: Thank you so much for the time and efforts spent on reviewing our\nmanuscript. We sincerely appreciate your positive and encouraging comments.\nAbstract\n1. Lines 36-38, \"Here, we developed a CRISPR/spCas9-based gene-editing strategy to\nprecisely excise the expanded GGC repeats in NOTCH2NL...\" To be specific, it should\nbe NOTCH2NLC, not NOTCH2NL.\nAuthor response: Thank you for pointing this out. The correct gene name should be\nNOTCH2NLC, and we have made the corresponding revision in the text.\nIntroduction\n1. Lines 89–97, the description of the function of the NOTCH2NL family genes can be\ndeveloped more here. Specifically, it is informative to clarify why preserving\nNOTCH2NL family genes is important—e.g., which one(s) are involved in cortical\ndevelopment and must remain unaltered to avoid developmental deficits.”\nAuthor response: Thank you for the suggestions. The NOTCH2NL gene family plays\ncritical roles in human brain development and homeostasis. Specifically, NOTCH2NLA,\nNOTCH2NLB, and NOTCH2NLC are expressed in cortical progenitors, where they\nenhance self-renewal and delay premature differentiation by potentiating canonical\nNotch signaling, thereby contributing to the evolutionary expansion of the human\ncerebral cortex. Abnormal dosage of NOTCH2NL genes is associated with the 1q21.1\ndeletion/duplication syndrome. Deletions are linked to microcephaly or cortical\nmalformations, while duplications correlate with macrocephaly or autism spectrum\ndisorders1-3. Thus, it is important to preserve the functions of NOTCH2NL family genes.\nWe have incorporated this information in the revised text as follows:\nThe human-specific NOTCH2NL (NOTCH2 N-terminal like) gene family, which\nincludes NOTCH2NLA, NOTCH2NLB, NOTCH2NLC and NOTCH2NLR, shares high\nsequence homology and plays critical roles in human brain development and\nhomeostasis3-5. Specifically, NOTCH2NLA, NOTCH2NLB, and NOTCH2NLC are\nexpressed in cortical progenitors, where they enhance self-renewal and delay premature\ndifferentiation by potentiating canonical Notch signaling, thereby contributing to the\nevolutionary expansion of the human cerebral cortex. Abnormal dosage of NOTCH2NL\ngenes has been linked to neurodevelopmental disorders, including microcephaly or\ncortical malformations upon loss and macrocephaly or autism spectrum disorders upon\nduplication. Therefore, it is essential to specifically target the GGC repeat expansion\nwithin NOTCH2NLC while preserving the normal expression and function of the\nNOTCH2NL family. However, the near-identical sequence flanking the GGC repeats\n(<2% divergence) among these homologous genes, combined with the large size and\nhigh GC content of the expanded repeats, pose significant challenges for precise and\nefficient gene editing.\nResults and Figures\n1. Line 123, one of the downstream targeted sequences do not match between the main\ntext (864), Fig.1C (864), Fig.S1B (866) and Table.S1 (866). In addition, please describe\nwhat the numbers in the names of gRNAs mean. If their names are from their sequence,\ndoes the number mean the beginning of the sequences or the end of the sequences or\nsomething else. Please also confirm if their names match with their actual number in\ntheir sequences. For example, it is puzzling what 702 and 703 are named after since\nthey are not 1 nucleotide apart, which could mis-lead authors intention to readers. Once\nauthors clarify/confirm the nomenclatures, the labels in the cartoon of Figure S1B,\nmain text and the Figure Legends all need to be rechecked and revised accordingly.\nAuthor response: Thank you for your careful scrutiny of our manuscript. The\ndesignation of sgRNA864 was a typographical error and should have been sgRNA866.\nWe apologize for the confusion caused by this misleading nomenclature. The original\nnames of the sgRNAs were based on their physical cleavage positions on chromosome\n1 of the GRCh38/hg38 reference genome. To avoid ambiguity, we have now renamed\nthem sequentially as sgRNA1-8. In addition, we provide a new table summarizing\ndetailed information for each sgRNA, including genomic coordinates, strand\norientation, and other relevant features. We have carefully rechecked and revised the\nlabels in the main text, figure legends, and figures according to the corrected\nnomenclature.\nThe new Supplementary Table 1 is shown below:\nOn-target\nOff-target\nName Cut position Strand Sequence PAM efficiency\nprofile\n(%)\nTGCTTCGGACCGTAGCG NOTCH2NLC\nsgRNA1 149390717 + GGG 76\nCCA -specific\nGTGCTTCGGACCGTAGC\nsgRNA2 149390716 + AGG 46 Not evaluated\nGCC\nGGCGCTACGGTCCGAAG\nsgRNA3 149390702 - AGG 63 Not evaluated\nCAC\nAGGCATTTGCGCCTGTG\nsgRNA4 149390702 + CGG 60 Not evaluated\nCTT\nCGCCCTGCGCCGCTCTGC NOTCH2NLC\nsgRNA5 149390874 + TGG 56\nTG -specific\nGCCCACAGCAGAGCGGC NOTCH2NLC\nsgRNA6 149390865 - GGG 57\nGCA -specific\nCGCCCACAGCAGAGCGG\nsgRNA7 149390866 - AGG 43 Not evaluated\nCGC\nCACAGCAGAGCGGCGCA\nsgRNA8 149390862 - CGG 39 Not evaluated\nGGG\n2. Lines 126-130, “First, the gene-editing efficiency of each sgRNA was evaluated in\nhuman cell lines with normal GGC repeats (HEK293 cells). Forty-eight hours after co-\ntransfection with Cas9 and sgRNA plasmids (Fig.S1C) at a Cas9-to-sgRNA ratio of\n4:1…” what is the rationale behind choosing 4:1? If there is a reference that authors\nfollowed, please cite the paper. Otherwise, authors need to briefly clarify why 4:1 was\nchosen to begin with.\nAuthor response: The 4:1 Cas9-to-sgRNA ratio was selected for preliminary screen\nbased on our laboratory's prior experience with genome editing in repeat expansion\ndisorders. We have now cited a relevant reference supporting this choice6, as it\nrepresents a standard practice in our lab for preliminary efficiency testing of different\nsgRNAs.\nLater (Line 145), it is mentioned that “the most effective ratio was found to be 1:1”.\nAny reason that authors chose to test 1:1, 2:1, 4:1 instead of the opposite ratios for\nCas9 to sgRNA (e.g., more sgRNA to Cas9 ratio such as 1:2 or 1:4)? If authors found that\n1:1 ratio is most effective among tested ratios, one could think that 1:2 could be even\nmore effective if tested. Please clarify it.\nAuthor response: We appreciate your insightful comment. After identifying the\neffective individual sgRNA, we subsequently examined different combinations at\nvarying ratios. Our experimental results (Supplementary Fig. 2C) demonstrated that,\namong the three ratios we tested (4:1, 2:1, and 1:1), the 1:1 ratio was the most effective\nwhen targeting both upstream and downstream sites simultaneously. We did not test\nlower Cas9 to sgRNA ratio (such as 1:2 or 1:4) for the following reasons. First, the\ncoding sequence of Cas9 (~4.2kb) is much larger than that of sgRNA (<0.1kb).\nTherefore, under equivalent transfection conditions, a given mass of plasmid DNA may\nyield a substantially higher molar amount of sgRNA than Cas9. Second, previous\nstudies have suggested that an excess of sgRNA may increase off-target effects7-9.\n3. Human phenotypes: PolyG aggregation was assessed in both HEK293T cells and\nmouse tissues, yet it was not evaluated in patient-derived human cells. Given its\npathological relevance and the consistency of analysis across models, there appears to\nbe no justification for omitting polyG detection in human fibroblasts. Including this\ndata would strengthen the translational relevance of the findings and enable a more\ndirect comparison across all experimental systems.\nAuthor response: Thank you for the invaluable suggestion. To date, it remains unclear\nwhether there are intranuclear inclusions in cultured skin fibroblasts of NIID patient.\nTo follow this advice, we examined polyG inclusions in cultured fibroblasts derived\nfrom a NIID patient (carrying 97/22 GGC repeats in NOTCH2NLC) and a health control\n(with 16/20 GGC repeats). Two antibodies were employed: NOTCH2NLC-polyG (a\nspecific antibody verified in our previous study10) and P62 (widely used in clinical\npathological diagnosis) were used. We did not observe the specific presence of polyG\npuncta in the NIID patient-derived fibroblasts. It is possible that the polyG inclusions\nare absent in the cultured skin fibroblasts under the in vitro condition. Alternatively, the\navailable antibody we used may not specifically or sensitively label polyG aggregates\nin human fibroblasts due to cell-type specific modifications. Because of these possible\nreasons, we were unable to confidently confirm the presence of polyG inclusions in\nfibroblasts and did not proceed with testing polyG inclusions in cultured human\nfibroblasts. Please see the following figures that are not included in the revision.\n[Figure Redacted]\n4. Figure 2: Are the GFP level alterations demonstrated in this Figure solely due to the\nGGC shortening or could that be also partially due to the frameshift location after the\nshortening and leading GFP not be able to be expressed? Please clarify this in the main\ntext. In addition, can the labels of poly G be modified to ‘GFP: PolyG’ instead of\n‘PolyG’ as authors did in their previous publication (Sci Adv, 2022) to\navoid any misleading/overstatement?\nAuthor response: We appreciate your thoughtful comment. We agree that the lack of\nGFP expression in the stable cells could result either from GGC repeat deletion or from\ndisruption by an out-frame shift. To distinguish these possibilities, we designed a pair\nof primers: the forward primer was located upstream of the upstream targeting region,\nand the reverse primer was within the GFP sequence. This primer pair specifically\namplifies the exogenous NOTCH2NLC-98GGC-GFP fragment without amplifying the\nendogenous NOTCH2NLC gene.\nAgarose gel electrophoresis showed that PCR amplification from the genomic DNA of\nthe 98polyG stable cell line treated with Cas9 and control sgRNA yielded a single 587\nbp band, corresponding to the allele carrying 98 GGC repeats. In contrast, cells treated\nwith Cas9 and sgRNA1+6 exhibited additional 187 bp and 350 bp bands\n(Supplementary Fig. 3A). Subsequent TA cloning and Sanger sequencing revealed that\nthe 187 bp product resulted from complete excision of the sequence between the two\nsgRNA target sites, whereas the 350 bp product contained approximately 22 GGC\nrepeats (within the normal range) and maintained the in-frame GFP fusion\n(Supplementary Fig. 3B-C). Sequencing of the 587 bp band confirmed that none of the\n15 analyzed TA clones exhibited a frameshift mutation (Supplementary Fig. 3D).\nCollectively, these findings indicate that the reduction of polyG-GFP protein levels in\nthe stable cell line primarily results from deletion or contraction of the expanded GGC\nrepeats rather than frameshift mutations. The GGC deletion efficiency was\napproximately 60% (Supplementary Fig. 3E).\nAdditionally, we updated the label of polyG to “GFP: PolyG” in Figure 2 to maintain\nconsistency with our previous publication and avoid potential misinterpretation or\noverstatement.\nSupplementary Figure 4\n5. Figure 3: The numerical values presented in the text appear consistent overall;\nhowever, in Figure 3H and 3I, the sum of individual dots within certain conditions does\nnot exactly match the corresponding group totals. For example, in Figure 3I, the sum\nof two data points in the “NIID down” group, it is 341 + 903 = 1244, and in the “GGC-\nrep down” group, it is 323 + 1394 = 1717, which aligns well with the indicated totals.\nHowever, in other samples or in Figure 3H, the individual data points do not sum\nprecisely to the indicated total. Could the authors clarify whether the plotted values\nrepresent raw counts, normalized data, or are derived from averages across replicates?\nPlease revise accordingly and as needed.\nAuthor response: Thank you for carefully reviewing our data presentation. Upon re-\nexamination, we confirm that the values plotted in these Upset plot figures represent\nthe number of differentially expressed genes (DEGs) identified from the comparison\ngroups shown in Figure 3E-G. These values are not raw sequencing counts (e.g., raw\nreads, FPKM, or TPM), but rather the results of bioinformatic analysis based on three\nbiological replicates per group and defined using the thresholds: |log2(fold change)| ≥\n2 and FDR < 0.05.\nThe discrepancies between the sum of individual data and the indicatedgroup totals\nwere due to incomplete data display. In original Figure 3H, 16 genes upregulated both\nin the GGC-deletion-NPC and NIID NPC, and 29 genes downregulated both in the\nGGC-deletion-NPC and NIID NPC were not shown. In original Figure 3I, 18 genes\nupregulated both in the GGC-repair-NPC and NIID NPC were not shown. We have\nsupplemented the omitted data in the revised figures. The individual data now\naccurately sum up to the indicated group totals, ensuring the consistency and accuracy\nof the data presentation. The updated Figure 3H and 3I are shown below.\nUpdated Figure 5H-I\nIn addition, in original Supplementary Figure 10G, one gene consistently upregulated\nand three genes consistently downregulated in both the treat and untreated group were\nnot shown. We have supplemented the omitted data in the revised figure. The individual\ndata now accurately sum up to the indicated group totals. The updated Supplementary\nFigure 12G is shown below.\nUpdated Supplementary Figure 12G\n6. Figure 3: Can authors indicate what color means what in their Figure legends and\ncan authors consider to use consistent color code for Figure 3I and 3H? For example,\nyou can match the color of NIID down and NIID up between Figure 3I and 3H.\nAuthor response: Thank you for the invaluable suggestion. We have updated the figure\nlegends to clearly indicate what each color represents. The dark blue and dark red\nrectangles indicated genes down-regulated and up-regulated in the unedited NIID NPC,\nrespectively. The light blue and pink rectangles indicated genes down-regulated and up-\nregulated in the edited NIID NPC, respectively. Additionally, we have made the color\ncode consistent for Figure 3I and 3H, specifically matching the colors for NIID down\nand NIID up in these two figures.\n7. Figure 4 and Figure S8: Authors should include age-matched wild-type control (or\nnon-disease control group) to demonstrate the phenotype of NIID mice as their control\ngroup (as authors did in Figure 5B-5I). Without comparing with WT mice, authors\ncannot conclude what authors have demonstrated in treated NIID mice as they rescued\nthe neuropathological phenotypes.\nAdditionally, what are the differences between Figure 4 and Figure S8? Can authors be\nclear about their description on Figure S8 if there is any difference from Figure 4? Even\nif authors wanted to demonstrate Figure S8 as another representative Figure to support\nFigure 4, it is unclear if the Figure S8 was representative images as a part of the\nquantification result presented in Figure 4 or if Figure S8 was an independent\nexperiment. If Figure S8 was an independent experiment, please have their quantified\nresult as a graph to support author’s conclusion about Figure S8. If not, please clarify\nin the main text and in the Figure legends.\nAuthor response: Thank you for your thoughtful comments. The original Figure S8\nconsists of the same cohort of mice as Figure 4, but shown at lower magnifications.\nSince these images did not provide additional information, we have removed it in the\nrevision.\nThe experiment in Figure 4 was designed as an initial in vivo test of whether localized\nknockout of the GGC repeat could ameliorate the pathological phenotype, before\nproceeding with retro-orbital venous injection. To minimize inter-individual\nvariability, therapeutic and control AAV were injected into the left and right striatum\nof the same NIID mouse, respectively. As you pointed out, this experiment alone does\nnot allow us to fully quantify the extent of recovery.\nFollowing your suggestion, we have now included a new Supplementary Figure 10 with\nage-matched non-disease control group. This new figure clearly demonstrates the\npathological hallmarks of NIID mice and shows that gene therapy markedly\nameliorated these abnormalities. Specifically, polyG inclusions were almost completely\ncleared to levels comparable to the non-disease controls, whereas NeuN expression and\ngliosis exhibited substantial but partial recovery.\nNew Supplementary Figure 10\n8. Figure 5J: The correlation between zsGreen expression, polyG reduction, and\nGFAP/NeuN changes appears inconsistent, making it difficult to link editing efficiency\nwith phenotypic rescue. For example, second sample of Treated NIID mice has higher\nexpression levels than other two mice, but their GFAP or NeuN levels looks fairly\nsimilar to each other between all 3 mice compared to how different they are from the\nsecond mouse. Authors need to clarify how or whether they normalized their data to the\nlevels of zsGreen. Comparing samples between mice which demonstrate comparable\nzsGreen expression levels would improve data interpretation and strengthen\nconclusions.\nAuthor response: Thank you for the invaluable suggestion. In the original Figure 5J,\npolyG, GFAP and NeuN protein levels were normalized to vinculin rather than zsGreen.\nWe agree that comparing mice with comparable zsGreen expression levels would\nimprove the interpretation. Accordingly, we reanalyzed data by including additional\nmice with comparable zsGreen expression levels. The updated Western blot results\ndemonstrated that, compared to the untreated NIID mice, treated NIID mice showed\nsignificantly reduced protein levels of polyG and GFAP, and increased NeuN levels in\nthe cortex. Notably, GFAP and NeuN expression in treated NIID mice were largely\nrestored to the levels in control mice (new Fig.5J-K). We acknowledge that some\nvariability in zsGreen expression was still observed across groups, which may reflect\ndifferences in AAV delivery efficiency among individual mice. Nonetheless, the\nconsistent directionality of changes in polyG, GFAP, and NeuN strongly supports our\nconclusion that the CRISPR/Cas9 editing system effectively mitigates\nneurodegenerative phenotypes in NIID mice.\nThe updated results are shown below (The treated group is NIID mice injected with\nAAV-Cas9 and AAV-sgRNA1+6; the untreated group is NIID mice injected with AAV-\nCas9 and AAV-control sgRNA; the control group is littermate control mice injected\nwith AAV-Cas9 and AAV-sgRNA).\nUpdated Figure 5J-K\n9. Figure 5K lacks a non-disease control group, making it difficult to evaluate the full\nextent of pathological elevation or rescue. The authors should include age-matched\nwild-type control group to establish a baseline and demonstrate whether treatment\nrestores polyG to the physiological levels.\nAuthor response: Thank you for this insightful suggestion. We have now included an\nage-matched non-disease control group in the revised Figure 5J-K (see figure above).\nBy adding this group, we were able to establish a clear baseline for the normal levels\nof polyG, GFAP, and NeuN. Our results showed that therapeutic AAV treatment in NIID\nmice restored polyG aggregation, GFAP and NeuN to the levels in non-disease control\nmice.\n10. Figure S1A: The second ‘Upstream mismatch sequences’ should be revised to be\n‘Downstream mismatch sequences’.\nAuthor response: Thank you for your careful scrutiny of our manuscript. We have\nrevised the second “Upstream mismatch sequences” in Figure S1A to “Downstream\nmismatch sequences” as suggested.\n11. Figure S3B: The X-axis labels indicate Untreated vs Treated. However, the\ndescription is missing in the Figure and Figure Legends about what they were treated\nwith. More detail information about Figure S3B needs to be added to the Figure\nLegends. In addition, the X-axis labels need to be revised to make it clearer and\nconsistent to other labels.\nAuthor response: Thank you for your careful scrutiny of our manuscript. We have added\ndetailed information about what the samples were treated with in the Figure Legends\nof Figure S3B. Also, we have revised the X-axis labels to make them clearer and\nconsistent with other labels.\n12. Figure S4: The order in the main text and the Figure is RP-PCR then GC-PCR,\nwhich doesn’t match with the Figure Legend (GC-PCR then RP-PCR). Please revise\nthe Figure Legend accordingly to match with other part of the manuscript.\nAuthor response: Thank you for your careful scrutiny of our manuscript. We have\nrevised the Figure Legend of Figure S4 to match the order of RP-PCR then GC-PCR,\nwhich is consistent with the main text and the Figure.\n13. Figure S10A: Y-axis label is missing from the graph.\nAuthor response: Thank you for your careful scrutiny of our manuscript. We have\nadded the Y-axis label to the graph in Figure S10A.\nMethods\n1. For animal study, were both sexes used, or only male or only female? If so, can\nauthors describe it clearly in the method section and justify the reason why they chose\nthose genders.\nAuthor response: In this study, both male and female mice were used in all experiments.\nWe did not observe sex-dependent differences in our analyses, which is consistent with\nprevious reports showing no significant differences in pathological or behavioral\nphenotypes between males and females10.\n2. Antibody information: It is better to indicate dilution ratios for primary and secondary\nantibodies to increase reproducibility of the study.\nAuthor response: We have now added dilution ratios in the method section for all\nprimary and secondary antibodies in the Methods section to improve the\nreproducibility of our study.\n3. Were any mortality or adverse events occurred during stereotaxic surgery or during\nthe behavior with animals who went through surgeries? If so, please describe it in the\nmanuscript.\nAuthor response: During stereotaxic surgery, no significant adverse events were\nobserved in P8-P10 mice receiving intrastriatal stereotaxic injections. All stereotaxic\nsurgeries were completed successfully, and surgical incisions healed satisfactorily\nwithout adverse events. The injected mice survived and underwent subsequent\npathological analyses without complications.\nFor retro-orbital venous injection, sixty-four neonatal P1-P2 pups were injected with\nAAV and less than ten pups died, likely due to poor rewarming after hypothermic\nanesthesia or cannibalized by the dam. The remaining pups survived and successfully\nunderwent subsequent experimental procedures.\nWe have included this information in Methods section.\n4. Information of patient age/sex and number of clones analyzed would strengthen rigor.\nPlease describe them as much as authors can.\nAuthor response: Thank you for the suggestion. The patient was a 57-year-old male\nwho has presented with bradykinesia and tremors in bilateral upper extremities for over\nten years. Physical examination revealed increased muscle tone in all four extremities\nand action tremors in bilateral upper extremities. Cognitive assessment yielded an\nMMSE score of 28 and a MoCA score of 27. The patient declined skin biopsy. Genetic\ntesting indicated a NOTCH2NLC gene carrying 113 and 17 GGC repeats. In addition,\nwe analyzed ninety iPSC clones in total, with four clones with the GGC repeats deleted\nor repaired.\nWe have added this information in the revised main text.\nReviewer #2 (Remarks to the Author):\nIn this work, Xie et al. have used a CRISPR/Cas9-based gene-editing approach to\nremove the expanded GGC repeats in the NOTCH2NLC gene, which is the known\ngenetic cause of neuronal intranuclear inclusion disease (NIID). To prevent off-target\neffects in homologous genes, the authors created and verified dual sgRNAs that flank\nthe repeat region. HEK293 cells, NIID patient-derived iPSCs, and a transgenic mouse\nmodel of NIID were used to validate the editing technique. The findings demonstrated\nthat the expansion of the repeat was efficiently removed, that pathological polyG\naggregates were reduced, that transcriptomic profiles were restored, and that the\nneurobehavioral phenotypes in mice were reversed. The work has great clinical\ntranslation potential and is backed by lots of phenotypic and molecular data. The\nmanuscript is strong in several ways. It presents a very specific, cleverly designed gene-\nediting technique and has been proven to work in a variety of biological systems. The\nfindings' robustness and translational potential are enhanced by the use of in vitro, ex\nvivo, and in vivo models. The paper offers a comprehensive examination that includes\nbehavioral evaluations in animal models, transcriptome rescue, and off-target\nconsequences. Furthermore, the work addresses important translational challenges like\nimmune response and vector administration within a therapeutically relevant\nframework. Overall the study is well planned and carried out. The main conclusions are\nsound, novel, and I think that this work is of high interest to the fields of genome editing\nand neurogenetics.\nAuthor response: Thank you very much for your thoughtful and encouraging comments.\nWe greatly appreciate your recognition of the novelty and translational relevance of our\nstudy, which motivates us to further pursue this line of research.\nI only have a few suggestions/remarks. First, only the top 14 projected loci are included\nin the off-target prediction. This could be improved by more thorough genome-wide\noff-target studies, such GUIDE-seq or CIRCLE-seq. Maybe the authors could discuss\nthis.\nAuthor response: We appreciate your insightful suggestion regarding off-target analysis\nand fully agree that unbiased genome-wide approaches are critical for assessing\ngenome-editing safety. CIRCLE-seq and GUIDE-seq are two widely adopted methods\nwith distinct advantages. CIRCLE-seq offers ultra-sensitive in vitro detection of\npotential off-target sites and is particularly valuable for sgRNA pre-screening11.\nHowever, because it is performed on naked DNA, it may overestimate off-target activity\nand does not reflect chromatin context. GUIDE-seq, by contrast, captures off-target\nevents in the native cellular environment, providing high sensitivity and clinical\nrelevance12 . Nevertheless, it requires specialized expertise and optimized protocols that\nwere not available to us in the present study. For these reasons, we did not apply\nCIRCLE-seq or GUIDE-seq here, but we acknowledge their importance and plan to\nincorporate these unbiased methods in future work.\nTo improve off-target assays, we combined targeted deep-seq and WGS to evaluate\npotential off-target activity in the revision. For deep-seq, we increased biological\nreplicates and extended coverage to the top 20 predicted sites, and consistently\nconfirmed that editing was restricted to NOTCH2NLC (revised Fig. 3A and new\nSupplementary Fig. 8A-B).\nNew Supplementary Figure 8\nFurthermore, we performed 50× WGS to assess overall genomic integrity and potential\noff-target mutations. WGS is considered a comprehensive approach for identifying\nSNVs, indels, CNVs, and structural variants13. Our results revealed comparable and\nnegligible extent of single-nucleotide variants (SNVs), structure variants (SVs) and\nindels between edited iPSCs and unedited iPSC (new Supplementary Fig. 8C). In\naddition, read-depth analysis of the additional 30 predicted off-target sites also showed\nno significant differences between groups (new Supplementary Fig.8D-E), further\nsupporting the specificity of our editing strategy.\nWhile the sensitivity of WGS for detecting extremely rare off-target events is generally\nlower than that of CIRCLE-seq or GUIDE-seq, the combination of targeted deep-seq\nand WGS offered complementary advantages, enabling a comprehensive assessment of\nediting specificity in this study. Incorporating unbiased, genome-wide approaches such\nas GUIDE-seq in future work will be essential for comprehensive off-target assessment\nprior to clinical translation.\nSecond, although the mouse model exhibits important characteristics, its limitations in\nreplicating the human disease should be discussed in more detail.\nAuthor response: Thank you for your invaluable comment. The transgenic NIID mouse\nmodel used in this study recapitulates several key pathological features of NIID,\nincluding polyG aggregates, neuroinflammation, and motor deficits10, making it an\nimportant tool for evaluating therapeutic efficacy. However, this model also has some\nlimitations.\nOne major limitation is its rapid disease progression: pathological and behavioral\nabnormalities emerge around 40 days of age, and the animals typically die prematurely\nby around two months10. This is in sharp contrast to human patients, who usually\ndevelop symptoms between 30 and 60 years of age and may survive for decades after\nonset14. This discrepancy may stem from the fact that the NOTCH2NLC gene is human-\nspecific, and its expression in rodent models may exacerbate the toxicity of expanded\nGGC repeats and accelerate disease progression due to the absence of native regulatory\npathways in mice.\nAnother limitation is that the current mouse model fails to reproduce key magnetic\nresonance imaging (MRI) features characteristic of NIID, such as hyperintensity signals\nalong the corticomedullary junction on diffusion-weighted imaging14,15. This difference\nmay be attributed to the simpler white matter architecture and lower glial cell density\nin mouse brains compared with humans. In contrast, large-animal models, particularly\nnon-human primates, may better recapitulate these MRI feature owing to their closer\ngenetic, physiological, and neuroanatomical similarities to humans, as well as their\nhigher proportion of glial cells16. Therefore, the development of large-animal NIID\nmodels, in combination with advanced neuroimaging approaches, represents a\npromising strategy for more faithfully modeling human disease and accelerating\ntranslational research, particularly for evaluating the efficacy, safety, and delivery\nefficiency of gene-editing therapies in a system that more closely resembles the human\nbrain.\nWe have incorporated this information in revised Discussion.\nThird, it would be good to include a summary table for sgRNAs, editing efficiencies,\nand off-target profiles.\nAuthor response: We have added a summary table including sgRNAs sequences, cutting\nposition, editing efficiencies, and off-target profiles in the revised supplemental files.\nIn addition, we have renamed the sgRNAs to avoid ambiguity.\nThe new Supplementary Table 1 was shown as below:\nOn-target\nOff-target\nName Cut position Strand Sequence PAM efficiency\nprofile\n(%)\nTGCTTCGGACCGTAGCG NOTCH2NLC\nsgRNA1 149390717 + GGG 76\nCCA -specific\nGTGCTTCGGACCGTAGC\nsgRNA2 149390716 + AGG 46 Not evaluated\nGCC\nGGCGCTACGGTCCGAAG\nsgRNA3 149390702 - AGG 63 Not evaluated\nCAC\nAGGCATTTGCGCCTGTG\nsgRNA4 149390702 + CGG 60 Not evaluated\nCTT\nCGCCCTGCGCCGCTCTGC NOTCH2NLC\nsgRNA5 149390874 + TGG 56\nTG -specific\nGCCCACAGCAGAGCGGC NOTCH2NLC\nsgRNA6 149390865 - GGG 57\nGCA -specific\nCGCCCACAGCAGAGCGG\nsgRNA7 149390866 - AGG 43 Not evaluated\nCGC\nCACAGCAGAGCGGCGCA\nsgRNA8 149390862 - CGG 39 Not evaluated\nGGG\nIn summary, my recommendation is accept with minor revisions.\nAuthor response: Thank you very much for your positive evaluation and\nrecommendation. We sincerely appreciate your encouraging comments.\nReviewer #3 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports.\nThis is part of the Nature Communications initiative to facilitate training in peer review\nand to provide appropriate recognition for Early Career Researchers who co-review\nmanuscripts.\nAuthor response: We sincerely appreciate your positive comments, careful review, and\nvaluable suggestions, which have been invaluable in refining our manuscript.\nReference\n1. Fiddes, I.T. et al. Human-Specific NOTCH2NL Genes Affect Notch Signaling\nand Cortical Neurogenesis. Cell 173, 1356-1369.e22 (2018).\n2. Suzuki, I.K. et al. Human-Specific NOTCH2NL Genes Expand Cortical\nNeurogenesis through Delta/Notch Regulation. Cell173, 1370-1384.e16 (2018).\n3. Florio, M. et al. Evolution and cell-type specificity of human-specific genes\npreferentially expressed in progenitors of fetal neocortex. eLife7(2018).\n4. Fiddes, I.T. et al. Human-Specific NOTCH2NL Genes Affect Notch Signaling\nand Cortical Neurogenesis. Cell 173, 1356-+ (2018).\n5. Suzuki, I.K. et al. Human-Specific NOTCH2NL Genes Expand Cortical\nNeurogenesis through Delta/Notch Regulation. Cell 173, 1370-+ (2018).\n6. Yang, S. et al. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity\nin mouse model of Huntington's disease. J Clin Invest 127, 2719-2724 (2017).\n7. Kim, S., Kim, D., Cho, S.W., Kim, J. & Kim, J.-S. Highly efficient RNA-guided\ngenome editing in human cells via delivery of purified Cas9 ribonucleoproteins.\nGenome Research24, 1012-1019 (2014).\n8. Fu, Y. et al. High-frequency off-target mutagenesis induced by CRISPR-Cas\nnucleases in human cells. Nature Biotechnology31, 822-826 (2013).\n9. Hsu, P.D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases.\nNature Biotechnology31, 827-832 (2013).\n10. Liu, Q. et al. Expression of expanded GGC repeats within NOTCH2NLC causes\nbehavioral deficits and neurodegeneration in a mouse model of neuronal\nintranuclear inclusion disease. Sci Adv8, eadd6391 (2022).\n11. Tsai, S.Q. et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-\nwide CRISPR–Cas9 nuclease off-targets. Nature Methods 14, 607-614 (2017).\n12. Tsai, S.Q. et al. GUIDE-seq enables genome-wide profiling of off-target\ncleavage by CRISPR-Cas nucleases. Nature Biotechnology33, 187-197 (2015).\n13. Anderson, K.R. et al. CRISPR off-target analysis in genetically engineered rats\nand mice. Nature Methods 15, 512-514 (2018).\n14. Tian, Y. et al. Clinical features of NOTCH2NLC-related neuronal intranuclear\ninclusion disease. J Neurol Neurosurg Psychiatry 93, 1289-1298 (2022).\n15. Tai, H. et al. Clinical Features and Classification of Neuronal Intranuclear\nInclusion Disease. Neurol Genet 9, e200057 (2023).\n16. Yin, P., Li, S., Li, X.J. & Yang, W. New pathogenic insights from large animal\nmodels of neurodegenerative diseases. Protein Cell 13, 707-720 (2022).\nResponses to referees’ and editors’ comments\nWe sincerely thank the reviewers for their time, insightful comments, and constructive\nsuggestions, which have significantly improved the quality of our work.\nIn this version, we have thoroughly reviewed the manuscript and supplementary\nmaterials against the journal’s checklist and have implemented all necessary revisions\nto ensure full compliance (all changes are highlighted in blue in the manuscript). We\nbelieve the manuscript now fully meets the journal’s standards and hope it is deemed\nsuitable for publication.\nThe detailed responses are shown below."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Authors addressed all of my concerns. The readability and the solidity of the manuscript improved significantly.", "reviewer_2": "The authors have thoroughly addressed my comments and have significantly improved the manuscript through their revisions. I have no further suggestions and recommend the manuscript for publication.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for their positive feedback and for confirming that our revisions\nhave addressed their concerns and enhanced the manuscript's readability and solidity.\nWe thank the reviewer for their supportive comments and for recommending our\nmanuscript for publication. We are pleased that the revisions have successfully\naddressed all points raised.\nWe thank the reviewer for their time and contribution to the review of our manuscript."}]

Decision letter