Using CRISPR/Cas9 To Study How CTNNB1 Mutation Influences Neural Progenitor Cell Gene Expression And Proliferation

Autism spectrum disorder(ASD)is a range of conditions classified as neurodevelopmental disorders.It’s a subtype of generalized developmental disorders and affected more males.Symptoms are typically recognized at early age.Patients were diagnosed with problems in social communication and social interaction,restricted and repetitive patterns of behavior.Some patients might show talent at one aspect.ASD is estimated affected about 1%of people worldwide.No official report of exactly incidence rate in China.The specific risk factors of ASD have yet to be found,genetic and environment factors had been identified.And there is no cure of ASD,which made ASD became an issue for clinicians.Most ASD are polygenic genetic disease.Only few ASD patients had specific genes,such as FMR1 in fragile X syndrome,MECP2 in Rett syndrome.With the development of deep sequencing and case collection,more and more susceptibility genes were found,such as CHD8,SCN2A and SYNGAP1.Simons Foundation Autism Research Initiative(SFARI)built a dataset to collect ASD susceptibility genes and divided the genes into five categories.In 2016,Pro.Wynshaw-Boris found a stop-gain mutation at CTNNBI(chr3:41266229 C>C/T).CTNNB1 involved into the Wnt pathway,which regulated brain development and animal behavior.But up to now,no solid evidence illustrated the relationship between CTNNB1 gene and ASD related behavior alternation and brain over development.Thus,our study focused on to Human Induced Pluripotent Stem Cells(hiPSC)to study how CTNNB1 stop-gain mutation influences neural progenitor cell gene expression and proliferation.The study preceded followed these three parts:(1)Using CRISPR/Cas9 to create a stop-gain mutation in CTNNB1 in humaninduced pluripotent stem cell lines.(2)Induce gene edited hiPSCs into Neural Progenitor Cells(NPCs)(3)CTNNB1 stop-gain mutation influences gene expression,cell proliferationand Wnt pathway activityPart ⅠObjectiveUsing CRISPR/Cas9 gene editing system to create iPSC models for autism patients.Use whole genome sequencing(WGS)to validate CRISPR/Cas9 gene editing efficiency and off-target rate.DesignUtilize homologous recombination mediated CRISPR/Cas9 gene editing to generate iPSC lines with a stop-gain mutation in CTNNB1 that was previously described in an autistic patient.Validate the mutation with Taqman probe,Sanger sequencing and WGS.ResultsWith CRISPR/Cas9,we successfully generated iPSC lines with the desired stop-gain mutation in CTNNB1;Taqman probe analysis indicated 23.96%induction efficiency in control cell lines and 31.25%efficiency for correction in autistic cell lines.WGS validated the CTNNB1 stop-gain mutation generated in control cell lines,but determined genomic editing failed to correct the stop-gain mutation in the autistic cell line.There were no major off-target effects in all cell lines analyzed.However,WGS revealed many single nucleotide polymorphism in these cell lines.ConclusionHomologous recombination mediated CRISPR/Cas9 gene editing could be used to generate hiPSC lines with stop-gain mutations in CTNNB1.WGS can be applied to detect both on-and off-target effects.Generation of a stop-gain mutation in a control cell line was successful but correction of this mutation in an autistic cell line was unsuccessful.Additionally,the off-target rate of CRISPR/Cas9 gene editing system was extremely low.Part IIObjectiveCRISPR/Cas9 edited hiPSC lines were used to investigate NPC differentiation and address the following questions.First,can a single SMAD inhibition protocol differentiate hiPSCs into NPCs as in human embryonic development?Second,are there any other unique markers expressed during embryonic body formation?Third,do these NPCs express the dorsal NPC marker PAX6.DesignDesign a novel single SMAD inhibition protocol to differentiate hiPSCs into NPCs,and use RT-qPCR to validate the differentiation process follows previously identified principles of brain development.Immunofluorescence was also utilized to test whether dorsal NPC markers were expressed.ResultsSingle SMAD inhibitor differentiation protocol could produce PAX6 positive NPCs,and the development process was similar to human embryonic development.This protocol can also be utilized in hESCs,other hiPSC lines and mESCs.ConclusionSingle SMAD inhibition differentiation protocol can convert hiPSC into NPCs,which could be useful as a neurodevelopmental model.Part ⅢObjectiveTo study whether CTNNB1 haploinsufficiency is the cause of NPC over-proliferation and brain overgrowth in our autistic hiPSC line.DesignWe applied RT-qPCR,Western-blot,TOP-flash,cell proliferation assays,and RNA-seq to study the influence of CTNNB1 haploinsufficiency in NPCs.ResultsRT-qPCR revealed CTNNB1 haploinsufficiency decreased p-catenin expression,and RNA-seq results validated the relative reduction.However,the protein level did not change.TOP-flash indicated the CTNNB1 stop-gain mutation decreased activity of the Wnt pathway.RNA-seq also validated the down steam gene expression changes.The mutation did not alter cell proliferation and adhesion.ConclusionCTNNB1 haploinsufficiency altered β-catenin expression but not corresponding phenotypes such as cell proliferation and cell adhesion.NPC over-proliferation may be the result of multiple factors.