Efficient Manipulation Of Gene Dosage In Human IPSCs Using .

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PENEfficient manipulation of gene dosage in humaniPSCs using CRISPR/Cas9 nickases1234567890():,;Tao Ye 1,2,3,4, Yangyang Duan 1,2, Hayley W. S. Tsang1,2, He Xu1,2, Yuewen Chen1,3,4, Han Cao1,2,Yu Chen1,3,4, Amy K. Y. Fu1,2,3 & Nancy Y. Ip 1,2,3 The dysregulation of gene dosage due to duplication or haploinsufficiency is a major cause ofautosomal dominant diseases such as Alzheimer’s disease. However, there is currently norapid and efficient method for manipulating gene dosage in a human model system such ashuman induced pluripotent stem cells (iPSCs). Here, we demonstrate a simple and precisemethod to simultaneously generate iPSC lines with different gene dosages using paired Cas9nickases. We first generate a Cas9 nickase variant with broader protospacer-adjacent motifspecificity to expand the targetability of double-nicking–mediated genome editing. As aproof-of-concept study, we examine the gene dosage effects on an Alzheimer’s diseasepatient-derived iPSC line that carries three copies of APP (amyloid precursor protein). Thismethod enables the rapid and simultaneous generation of iPSC lines with monoallelic, biallelic, or triallelic knockout of APP. The cortical neurons generated from isogenically correctediPSCs exhibit gene dosage-dependent correction of disease-associated phenotypes ofamyloid-beta secretion and Tau hyperphosphorylation. Thus, the rapid generation of iPSCswith different gene dosages using our method described herein can be a useful model systemfor investigating disease mechanisms and therapeutic development.1 Divisionof Life Science, State Key Laboratory of Molecular Neuroscience, Center for Stem Cell Research, Molecular Neuroscience Center, The Hong KongUniversity of Science and Technology, Clear Water Bay, Hong Kong, China. 2 Hong Kong Center for Neurodegenerative Diseases, Hong Kong, China.3 Guangdong Provincial Key Laboratory of Brain Science, Disease and Drug Development, HKUST Shenzhen Research Institute; Shenzhen-Hong KongInstitute of Brain Science, 518057 Shenzhen, Guangdong, China. 4 The Brain Cognition and Brain Disease Institute, Shenzhen Institute of AdvancedTechnology, Chinese Academy of Sciences; Shenzhen-Hong Kong Institute of Brain Science, 518055 Shenzhen, Guangdong, China. email: boip@ust.hkCOMMUNICATIONS BIOLOGY (2021)4:195 https://doi.org/10.1038/s42003-021-01722-0 www.nature.com/commsbio1

ARTICLETCOMMUNICATIONS BIOLOGY https://doi.org/10.1038/s42003-021-01722-0he complexity of the human brain arises not only from thegenes it expresses but also from the precise regulation ofgene dosage and expression levels. Their dysregulation dueto duplication or haploinsufficiency is a major cause of autosomaldominant diseases, such as Alzheimer’s disease (AD)1,2. As themost common type of neurodegenerative dementia, AD is characterized by two pathological hallmarks: extracellular amyloidplaques largely comprising amyloid-beta (Aβ) peptides andintracellular neurofibrillary tangles rich in hyperphosphorylatedTau protein3. In AD, the duplication of specific genes, such asAPP (amyloid precursor protein) can cause rare early-onset formsof the disease termed “familial AD”4. Meanwhile, heterozygouspremature termination codon and noncoding variants in a singleallele of certain genes5,6 (e.g., ABCA7) confer a risk of late-onsetsporadic AD. Given that numerous promising therapeuticsshowed efficacy in animal models of AD but failed in clinicaltrials7, there is a pressing need to establish relevant model systems, such as human neurons that better mimic the pathophysiology of AD in humans.Technological developments have enabled the generation andsubsequent differentiation of human-induced pluripotent stemcells (iPSCs) into various brain cell types and brain organoids8.Thus, iPSC-based models with AD-causing mutations can recapitulate certain phenotypes of familial AD, including increasedAβ production, Tau hyperphosphorylation, endosomal abnormalities, and oxidative stress9–12. However, when studying genesand variants associated with sporadic AD that might have fewerrisk effects, interindividual genetic heterogeneity can hinder theaccurate analysis of disease phenotypes in vitro13. Therefore,genome-editing tools, such as the bacterial CRISPR (clusteredregularly interspaced short palindromic repeats)/Cas9 system,have great potential to generate isogenic iPSC lines that onlydiffer with respect to a specific gene or mutation of interest butare otherwise genetically identical14,15. Although heterozygousknockout more accurately models the partial reduction in geneexpression caused by noncoding variants, most sporadic ADgenes have not been studied in heterozygous-knockout iPSCbased models in parallel to their homozygous-knockout counterparts (summarized in Supplementary Table 1)16, partlybecause there is currently no rapid and efficient method tomanipulate gene dosage in iPSCs.RNA-guided Cas9 nuclease enables sequence-specific, doublestrand breaks by specifying a 20-nucleotide targeting sequencewithin its single-guide RNA (sgRNA) followed by a protospaceradjacent motif (PAM) sequence17. For the most commonly usedStreptococcus pyogenes Cas9 (“Cas9” hereafter), the required PAMsequence is NGG17. When targeting the exonic regions of a gene,Cas9-induced double-strand breaks can result in frameshiftmutations and gene knockout through DNA repair mediated bynon-homologous end-joining18. As Cas9-mediated gene knockout appears to be highly efficient and insensitive to gene copynumber19, Cas9 with a potent sgRNA tends to yield completeknockout of target genes, which can facilitate loss-of-functionstudies20–23 (summarized in Supplementary Table 2). However,given the considerable off-target effects of Cas9 nuclease24,25, theuse of paired Cas9 nickases (Cas9n) can significantly reduce suchoff-target effects26. As both nickases must be functional to generate two single-strand breaks in close proximity (i.e., 0–20nucleotides between two sgRNAs)27, the genome targetability ofCas9n is expected to be much lower than that of an individualCas9 nuclease, thus necessitating further optimization. Furthermore, the ability of paired nickases to manipulate gene dosage iniPSCs has not been evaluated at the single-cell-derived clone level.In this study, we expanded the genome-editing targetability ofpaired nickases by generating a Cas9nVQR variant (D1135V/R1335Q/T1337R) with an NGA PAM. In addition, by using an2AD iPSC line carrying APP duplication (“APP cells” hereafter),we used paired Cas9nVQR for the one-step generation of isogeniciPSC lines with monoallelic, biallelic, or triallelic knockout of thetarget gene; among them was an isogenically corrected line (“Isocells” hereafter) that had undergone monoallelic APP disruption(i.e., carrying two functional APP alleles) as an APP wild-typerevertant. After the iPSCs differentiated into cortical neurons,compared to APP neurons, Iso neurons exhibited genotypedependent rescue of Aβ production and Tau phosphorylation atThr231. Furthermore, transcriptomic profiling and functionalvalidation revealed a neuronal apoptotic pathway that is potentially implicated in APP dosage-dependent AD pathogenesis.Thus, our study demonstrates an efficient approach for rapidlymanipulating gene dosage in iPSCs with paired Cas9n, which willfacilitate the study of risk genes associated with human diseases.ResultsFootprint-free editing of APP copy number by paired Cas9nickases. To expand the genome-editing targetability of pairednickases, we generated a Cas9nVQR variant (D1135V/R1335Q/T1337R) with an NGA PAM28 (Supplementary Fig. 1a–d).Genome-wide coverage analysis demonstrated that Cas9n cantarget 58.1% of 3,209,286,105 sites in the human reference genome (GRCh38). Meanwhile, the Cas9nVQR variant developed inthis study can increase the targetability to 79.8% when usedalone and to 90.3% when used together with Cas9n; this expanded genome targetability translates into 695,373,139 and1,032,629,268 genomic sites, respectively, that are not targetableby Cas9n (Supplementary Fig. 1e, Supplementary Software 1). Toexamine the ability and flexibility of the Cas9nVQR variant tomanipulate gene copy number in iPSCs at the single-cell-derivedclone level, we utilized an AD iPSC line carrying APP duplication(i.e., APP cells)9 and targeted APP exon 16, which is inaccessibleto wild-type Cas9n (Fig. 1a). Accordingly, we designed a pair ofsgRNAs—designated sgRNA1 and sgRNA2—that targeted thislocus (Fig. 1a). To determine their genome-editing efficiency, wetransfected constructs that express Cas9nVQR and correspondingsgRNAs into HEK 293T cells. We utilized the endogenous EcoRIsite in the target sequence to rapidly screen the genome-editingevents. An EcoRI-resistant band at 1.1 kb indicated the disruptionof an endogenous EcoRI site and successful editing at the targetedlocus (Fig. 1b). Successful genome editing occurred when bothsgRNAs and Cas9nVQR were expressed in the HEK 293T cells,whereas no editing occurred when Cas9nVQR was expressedalone or with only one of the two sgRNAs (Fig. 1b).To establish the APP copy number-corrected isogenic line (i.e.,Iso cells), we first transfected APP iPSCs with Cas9nVQR-GFPconstructs expressing sgRNA1 and sgRNA2 and isolated GFPpositive cells by cell sorting. Next-generation deep sequencingdemonstrated an editing efficiency of 40 3.5% in iPSCs afterGFP sorting. We subsequently sorted cells for single-cloneexpansion and screened them by EcoRI digestion assay asdescribed above (Fig. 1c). Out of the 12 screened iPSC clones,four exhibited potential genome editing at the target region(Fig. 1d). We did not detect large deletions in the 5.2-kb PCRamplicons surrounding the edited region in these clones; wesubsequently genotyped them by Sanger sequencing and nextgeneration sequencing (Supplementary Fig. 2a). Accordingly, wegenerated an Iso line (i.e., a corrected line) with two copies ofAPP and lines with one or zero copies of APP (Fig. 1e,Supplementary Fig. 2b). Further array-based comparative genomic hybridization (CGH) assay showed that the edited iPSC lineswith monoallelic disruption still carried a 722-kb duplication onchromosome 21 like the parent APP duplication iPSC line,whereas the iPSC lines with biallelic or triallelic disruption didCOMMUNICATIONS BIOLOGY (2021)4:195 https://doi.org/10.1038/s42003-021-01722-0 www.nature.com/commsbio

COMMUNICATIONS BIOLOGY ig. 1 Footprint-free gene editing of APP copy number in human induced pluripotent stem cells by paired Cas9 nickases. a Design of a scarless,antibiotic marker-free, gene-editing strategy using a newly generated Cas9 nickase variant, Cas9nVQR (D1135V/R1335Q/T1337R), with paired singleguide RNAs (i.e., sgRNA1 and sgRNA2) that target APP exon 16 in neurons with APP duplication. Red arrowheads indicate the nickase cleavage site, blueboxes indicate the exon, and the red line within the blue boxes indicates Cas9-induced insertion/deletion (indel) mutations. b Genome-editing efficiency ofCas9nVQR with paired sgRNAs in HEK 293T cells. Cas9nVQR with or without corresponding sgRNAs was transfected into HEK 293T cells. The EcoRIresistant band at 1.1 kb indicates successful editing. c Schematic diagram of genome editing in a human induced pluripotent stem cell (iPSC) line carryingduplication of the APP gene. d Screening of gene-edited iPSC clones by EcoRI digestion. e Specific deletion mutations detected in gene-edited iPSC clonesby Sanger sequencing and next-generation deep sequencing. The percentage of deep-sequencing reads for each iPSC clone indicates the number of APPcopies remaining intact or removed. One isogenic iPSC clone (Iso) had approximately one-third of 23-bp deletion reads and two-thirds of wild-typesequence reads, indicating that one copy of APP had been inactivated; therefore, it was used for subsequent experiments. PAM protospacer-adjacent motif.not (Supplementary Table 3, Supplementary Data 1). Theseresults collectively suggest that paired Cas9nVQR deletes theentire 722-kb duplicate region harboring the APP locus in iPSClines with biallelic or triallelic disruption, which is more than100-fold larger than that in the previous study ( 6 kb)27. We usedthis approach to replicate the efficient generation of the threegenotypes simultaneously, demonstrating its reproducibility(Supplementary Fig. 3). Therefore, the double-nicking methodflexibly and efficiently manipulated gene copy numbers.We subsequently examined whether the Iso iPSC linesmaintained the pluripotent characteristics of the parental APPiPSC line, specifically pluripotency marker expression and anormal karyotype9. Indeed, an Iso iPSC clone stained positive forpluripotent stem cell markers including OCT4, SSEA4, and TRA1-81 (Supplementary Fig. 4a) and had a normal karyotype uponCGH assay (Supplementary Fig. 4b).Impact of APP copy correction on amyloid- and Tauassociated pathologies. We subsequently induced the formationof cortical neurons from corresponding iPSC lines according tothe Ngn2 (neurogenin2)-mediated differentiation protocol15,29.After 28 days in vitro (DIV), APP, Iso, and nondemented control(NDC) iPSCs efficiently differentiated into forebrain corticalneurons that expressed the neuronal marker MAP2 (Fig. 2a). Inaddition, more than 90% of MAP2-positive neurons co-expressedthe layer II–IV neuronal marker Cux1 (Fig. 2b), suggesting that ahomogenous population of upper-layer cortical neurons had beengenerated. Moreover, RT-qPCR analysis indicated that the neurons induced from APP, Iso, and NDC iPSCs exhibited similarmRNA expression levels of NeuN (a mature neuronal marker),synaptophysin (a presynaptic marker), and PSD-95 (a postsynaptic marker) (Fig. 2c). These results indicate that theseneurons had a similar differentiation status.Neurons derived from APP iPSCs exhibit abnormal Aβ peptidesecretion and Tau hyperphosphorylation9. Indeed, compared toNDC neurons, APP neurons secreted significantly more Aβ38,Aβ40, and Aβ42 peptides into the medium. Meanwhile, subsequent correction of APP gene dosage in Iso iPSCs restored thenormal secretion levels of these Aβ peptides (Fig. 2d). Furthermore, in Iso iPSC-derived neurons, the APP protein leveldecreased close to that in neurons derived from the iPSCs ofNDCs, confirming the correction of APP copy number in IsoiPSCs (Fig. 2e, g).We subsequently examined whether APP gene copy numberaffected Tau phosphorylation in Ngn2-induced neurons fromAPP, Iso, and NDC iPSCs. Tau phosphorylated at Thr231, acomponent of paired helical filaments Tau, is an early marker ofAD pathology correlated with cognitive decline30. Compared toNDC neurons, APP neurons exhibited significantly higher Tauphosphorylation at Thr231, total Tau level, and pTau231/totalTau ratio, which is consistent with the previous studies9,11.Meanwhile, APP copy number correction in Iso neurons reducedthe levels of phosphorylated and total Tau (Fig. 2e–g), suggestingthat deleting the extra copy of APP restored Tau phosphorylationat Thr231, total Tau level, and the pTau231/total Tau ratio.COMMUNICATIONS BIOLOGY (2021)4:195 https://doi.org/10.1038/s42003-021-01722-0 www.nature.com/commsbio3

ARTICLECOMMUNICATIONS BIOLOGY https://doi.org/10.1038/s42003-021-01722-0Fig. 2 CRISPR/Cas9-mediated correction of APP copy number alleviates amyloid- and Tau-associated pathologies in induced pluripotent stem cellderived neurons. a, b Generation of a homogenous population of cortical neurons from nondemented control (NDC), APP duplication parent (APP), andAPP copy number-corrected isogenic (Iso) cell lines. a Neurons at 28 days in vitro (DIV) were stained for MAP2 (neuronal marker) and Cux1 (cortical layerII–IV marker). b Percentages of Cux1- and MAP2-positive neurons. c Neuronal differentiation status as assessed by qRT-PCR for MAP2, NeuN (matureneuron marker), Synaptophysin (presynaptic marker), and PSD-95 (postsynaptic marker). d Levels of secreted amyloid-beta (Aβ)38, Aβ40, and Aβ42 as wellas Aβ42/Aβ40 ratios in conditioned media from NDC, APP, and Iso neurons at 28 DIV. e Western blot analysis of full-length APP (FL-APP), phosphorylatedTau at Thr231 (pThr231 Tau), and total Tau in NDC, APP, and Iso neurons at 28 DIV. βIII-tubulin (βIII-tub) and GAPDH served as loading controls forneurons and total protein, respectively. f Immunocytochemical analysis of pThr231 Tau and doublecortin (DCX) in APP and Iso neurons at 28 DIV.g Quantification of APP/βIII-tub, pThr231 Tau/βIII-tub, total Tau/βIII-tub, and pThr231/total Tau ratios relative to those in NDC neurons. Values are mean SEM (n 4 independent biological replicates per cell line; *p 0.05, **p 0.01, or ***p 0.001 vs. APP neurons, Student’s t-test). Scale bars: 100 μm.4COMMUNICATIONS BIOLOGY (2021)4:195 https://doi.org/10.1038/s42003-021-01722-0 www.nature.com/commsbio

COMMUNICATIONS BIOLOGY https://doi.org/10.1038/s42003-021-01722-0These results are concordant with a recent study showingthat the pTau231/total Tau ratio is regulated through mechanisms involving cholesterol metabolism, cholesteryl esters, andproteasome degradation in iPSC-derived neurons with APPduplication31.Screening of pathways associated with APP gene dosage. Toscreen for the molecular pathways associated with APP genedosage, we profiled the transcriptomes of neurons from APP andIso iPSC lines. Differential gene expression analysis revealed 376differentially expressed genes (DEGs) between the APP and Isolines termed “isogenic DEGs.” Hierarchical clustering analysisrevealed that three batches of APP and Iso neurons clusteredtogether within each respective group (Fig. 3a). Among them,208 and 168 genes were up and downregulated, respectively, inAPP neurons compared to Iso neurons. This suggests that APPgene dosage broadly affects gene expression in a human diploidcell system. In parallel, differential gene expression analysisARTICLEbetween the APP and non-isogenic NDC lines yielded 2,158DEGs termed “non-isogenic DEGs” (including 1050 and 1108 upand downregulated genes, respectively). Venn diagram analysisof these two groups of DEGs revealed 207 common genes(Fig. 3b), which accounted for 55% of the isogenic DEGs (i.e.,APP vs. Iso) and less than 10% of non-isogenic DEGs (i.e., APPvs. NDC). Therefore, we analyzed these 207 common genes,which exhibited both APP-dependent effects and dysregulationin APP neurons.Functional annotation of these 207 common DEGs revealedseveral significantly enriched gene ontology categories including“regulation of transcription” (false discovery rate [FDR] 4.82E 4),“regulation of response to stimulus” (FDR 2.88E 2), “regulationof the Wnt signaling pathway” (FDR 3.84E 2), and “homophiliccell adhesion via plasma membrane adhesion molecules” (FDR 4.06E 2) (Fig. 3c). Subsequent analysis of the top genes using FDRfilters and visualization using volcano plots yielded candidates thatmight have functional implications in AD pathogenesis (Fig. 3d, e;Table 1). Therefore, we performed RT-qPCR to validate the top fourFig. 3 Transcriptomic profiling of isogenic induced pluripotent stem cell-derived neurons. a Hierarchical clustering analysis of the 376 differentiallyexpressed genes (DEGs) in APP duplication parent (APP), APP copy number-corrected isogenic (Iso), and nondemented control (NDC) neurons. Amongthem, 208 and 168 genes were up and downregulated, respectively, in APP neurons compared to Iso neurons (false discovery rate [FDR] 0.05, foldchange 1.5). b Venn diagram illustrating overlap between isogenic DEGs (APP vs. Iso neurons) and non-isogenic DEGs (APP vs. NDC neurons) (FDR 0.05, fold change 1.5). c Enriched Gene Ontology (GO) terms for the 207 common DEGs. Volcano plots showing the DEGs between the APP and NDCneurons (d) and between the Iso and APP neurons (e). Four selected genes upregulated in APP neurons compared to NDC neurons that were the topgenes downregulated in copy number-corrected Iso neurons compared to APP ne

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