{"paper_id":"e80a1b97-7cf6-4dc7-8978-b0aa374047d8","body_text":"1 \nCRISPRa-mediated activation of genes  associated with inherited retinal \ndystrophies in acutely isolated human cells for diagnostic purposes \n \nValentin J. Weber 1, A lice Reschigna2, Maximilian-J. Gerhardt 2, Klara S. \nHinrichsmeyer1, Dina Y. Otify 3, Thomas Heigl1, Frank Blaser 4, Isabelle Meneau 4, \nMartin Biel3, Stylianos Michalakis2# and Elvir Becirovic1# \n \n1Laboratory for Retinal Gene Therapy, Department of Ophthalmology, University \nHospital Zurich, University of Zurich, Zurich, Switzerland \n2Department of Ophthalmology, LMU University Hospital, LMU  Munich, Munich, \nGermany \n3Department of Pharmacy – Center for Drug Research, LMU Munich, Munich, \nGermany \n4Department of Ophthalmology, University Hospital Zurich, Zurich, Switzerland \n#corresponding author: elvir.becirovic@uzh.ch; michalakis@lmu.de \n \nRelated specialties: Genetics, Ophthalmology \nKeywords: Diagnostics, Genetic diseases, RNA processing \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n2 \nAbstract \nMany patients suffering from inherited diseases do not receive a genetic diagnosis and \nare therefore excluded as candidates for treatments, such as gene therapies. \nAnalyzing disease-related gene transcripts from patient cells would improve detection \nof mutations that have been missed or misinterpreted in terms of pathogenicity during \nroutine genome sequencing. However, the analysis of transcripts is complicated by the \nfact that a biopsy of the affected tissue is often not appropriate, and many disease -\nassociated genes are not expressed in tissues or cells that can be easily obtained from \npatients. Here, using CRISPR/Cas-mediated transcriptional activation (CRISPRa) we \ndeveloped a robust and efficient approach to activate genes in skin-derived fibroblasts \nand in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy \nindividuals. This approach was successfully applied to blood samples from patients \nwith inherited retinal dystrophies (IRD). We were able to efficiently activate several \nIRD-linked genes and detect the corresponding transcripts using different \ndiagnostically relevant methods such as RT-qPCR, RT-PCR and long- and short-read \nRNA sequencing. The detection and analysis of known and unknown mRNA isoforms \ndemonstrates the potential of CRISPRa-mediated transcriptional activation in PBMCs. \nThese results will contribute to ceasing the critical gap in the genetic diagnosis of \npatients with IRD or other inherited diseases.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n3 \nIntroduction \nEven in developed countries with modern molecular diagnostic equipment, up to 50% \nof patients with genetic diseases receive no or insufficient genetic diagnosis (1). This \nexcludes patients from established treatments and participation in clinical trials testing \nnovel therapies for the respective diseases. \nHigh-throughput next generation sequencing (NGS) technologies, such as whole \ngenome sequencing (WGS) or whole exome sequencing (WES) have facilitated the \ndiagnostics of genetic diseases. Both WGS and WES, howev er, have key limitations. \nWES does not cover the non-coding regions (e.g. introns, promoters or other regulatory \ntranscriptional elements), which are crucial for mRNA stability and processing. WGS \nis time and money -consuming, and the interpretation of the large amount of data \nobtained during this process is rather difficult  (2, 3). Even if potential disease-causing \nmutations can be identified in coding or non -coding regions of candidate genes using \nWGS, experimental validation of how these mutations influence the mRNA level is \ninevitable but laborious using currently available techniques (e.g. minigene assays (4) \nor patient -derived iPSC models). Single nucleotide variants can affect mRNA \nprocessing via different mechanisms. Among those, the most common one alters pre-\nmRNA splicing (5). Splice site mutations are typically classified as such using standard \nsplice prediction software (6, 7). Nevertheless, bioinformatic prediction of the effects of \nmutations i s often not reliable. Apart from false -positive results, splice prediction \nprograms also yield an uncertain number of false -negative records. Many false -\nnegative splicing mutations are classified as missense or silent variants located in \n(deep) exonic coding regions  (8, 9). However, such point mutations can also affect \nregulatory splice elements, ultimately leading to aberrant splicing, and should therefore \nbe routinely tested at the transcript level, preferably in patient cells (10). \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n4 \nTaken together, there is an unmet need for developing straightforward techniques \nfocusing on detection and investigation of disease -associated mutations at the \ntranscript level. The most convenient method to analyze the transcripts of the \ncorresponding genes is to use patients’ tissue samples. However, taking biopsies of \naffected tissues may often not be reasonable and many disease -relevant genes are \nnot expressed in cells which can be routinely obtained from the patients (e.g. blood \ncells or skin -derived fibroblasts). This applies in particular to IRDs, in which tissue \ncollection from the affected retina for diagnostic purposes is generally not justified due \nto the invasiveness of the procedure, risk of iatrogenic damage and potentially sight -\nthreatening complications. To circumvent these obstacles, we developed a CRISPRa-\nbased approach for transcriptional activation of genes in acutely isolated human cells \n(hereinafter referred to as CATALYTEC) to analyze single or multiple genes associated \nwith IRDs.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n5 \nResults \nTo provide proof of principle for CATALYTEC, we initially focused on ABCA4, RPE65, \nMYO7A and USH2A for several reasons: \ni) The individual genes are frequently mutated in the corresponding diseases: \nStargardt disease (STGD), Leber congenital amaurosis (LCA), Retinitis \npigmentosa (RP), and Usher syndrome (USH).  \nii) Three of the four genes (USH2A, MYO7A, and ABCA4) have a large gene \nbody, a condition that complicates the identification of potentially pathogenic \nmutations, particularly those located in non-coding regions. \niii) RPE65 offers high therapeutic relevance as RPE65 retinopathy can be \ntreated with the approved AAV gene supplementation therapy voretigene \nneparvovec. Improved genetic testing could help to confirm pathogenicity of \nnovel RPE65 variants of uncertain significance and thus qualify affected \npatients for treatment with the approved gene therapy. \nOur initial gene activation experiments were performed and optimized in HEK293T \ncells transfected with cassettes expressing nuclease deficient dCas9-VPR, one of the \nmost effective CRISPRa systems (11, 12), in combination with single guide (sg) RNAs \ntargeting the transcriptional start site (TSS) region of the  respective genes (Fig. 1A). \nWe were able to identify sgRNAs for efficient activation of each of these genes. \nMultiplexing experiments demonstrated that the combination of multiple sgRNA \ncassettes resulted in simultaneous activation of all genes without any substantial loss \nof activation efficiency (Fig. 1D) . Using RT -PCR, we could amplify all fragments \ncovering the entire coding region and parts of the untranslated regions (Fig. 1B+C , \nSuppl. Fig. 1). The identity of all bands was confirmed by Sanger sequencing. \nIn the first attempts to establish a robust and simple protocol for the transfection of \nPBMCs and skin fibroblasts from healthy individuals , we tested various commonly \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n6 \nused transfection techniques (e.g. calcium phosphate, lipofection and electroporation). \nFor most of these approaches, however, we observed no or weak expression of the \ntransfected GFP reporter gene. \nSince lentiviral vectors (LV) have been shown to efficiently transduce non-dividing cells \n(13), we investigated the efficacy of gene activation upon transduction with LVs. In our \nexperiments, using dual LV vectors, each expressing GFP or dsRed, we obtained high \nco-transduction efficiencies for human skin fibroblasts and PBMCs (Suppl. Fig. 2A, B). \nSurprisingly, the dual LV approach for gene transcriptional activation in transduced \nHEK293T, fibroblasts and PBMCs led to a relatively weak transcriptional activation of \nABCA4, despite numerous optimization steps (e.g. use of high multiplicity of infections \n(MOIs), different promoters etc., Suppl. Fig. 2C-F). These results suggest that dCas9-\nVPR delivered via LVs is substantially less efficient in activating target genes compared \nto delivery by plasmid transfection. To elucidate the causes for the low activ ity of \ndCas9-VPR in cells transduced with the corresponding LVs, we generated a HEK293T \ncell line stably expressing dCas9 -VPR through genomic integration of the \ncorresponding cassette (Suppl. Fig. 3A). After transduction of these cells with LVs \nexpressing only the sgRNAs targeting the individual genes, we detected a robust \ntranscriptional activation of ABCA4, MYO7A and USH2A. A similarly high activation \nwas also achieved in a multiplexing approach, in which all three genes were targeted \nsimultaneously (Suppl. Fig. 3B). In combination with the results shown in Suppl. Fig. \n2, this indicates that the efficiency of transcriptional activation is not compromised by \nLV-mediated delivery of sgRNAs, excluding a general impact of LVs on gene \nactivation. A possible explanation for our observation of low-level gene activation in all \ncell types upon LV-based administration of dCas9-VPR is that dCas9-VPR is not well \ntolerated by LV vectors due to its size (5.8 kb). To test this hypothesis, we used a \nshorter variant of this  CRISPRa system, dCas9 -VPRmini (14) (Suppl. Fig. 3C). \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n7 \nHowever, this approach also did not lead to increased activation efficiency compared \nto full-length dCas9-VPR (Suppl. Fig. 3D). \nFrom this set of unfavorable results, we concluded  that the LV -based approach in \ncombination with dCas9-VPR mediated transcriptional activation of genes is unsuitable \nfor diagnostic purposes. \nAchieving reasonable levels of transcriptional activation in PBMCs or skin fibroblasts \nfrom healthy individuals is a pr erequisite for establishing a robust protocol for \ndiagnostic purposes in patient cells. So far, although we initially obtained high \nactivation efficiencies for the individual IRD-linked genes in transfected HEK293T cells, \nnone of the previously described approaches has reached this goal. In another attempt \nto achieve this milestone, we used nucleofection for transferring sgRNA and transgene \nplasmids into our primary target cells. Nucleofection of a GFP -encoding cassette \nresulted in robust expression of this  reporter in both cell types (Suppl. Fig. 4A).  Next, \nwe nucleofected the dCas9-VPR expression cassette together with sgRNAs targeting \neither MYO7A, USH2A, ABCA4 or RPE65. Using RT -qPCR, we observed high \nactivation efficiencies for ABCA4 and RPE65 in both cell types. Additionally, we were \nable to detect the entire coding and untranslated regions using RT -PCR (Fig. 2A, B  \nand Suppl. Fig. 5 ). By comparison, we found that MYO7A is already endogenously \nexpressed in human skin fibroblasts and in PBMCs, respectively, in sufficient amounts \nto be detected by RT -PCR. Nevertheless, after transcriptional activation a moderate \nincrease in MYO7A expression was detectable via RT-qPCR (Suppl. Fig. 4B, C). For \nUSH2A, we could only detect parts covering exons 1 -7, 45-55 and the 3’ -UTR of the \ncorresponding mRNA (Suppl. Fig. 4D, E). \nFor RPE65 and ABCA4, in addition to the expected RT-PCR bands encoding the full-\nlength proteins, we detected several splice variants, most of which result ed in \nframeshifts and premature termination codons (PTC, Suppl. Tab. 1  + 2). The \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n8 \ncorresponding bands could represent alternative splicing that might differ in PBMCs \nand in the retina. \nTo test how the splice pattern observed in PBMCs compared to that of human retinal \ncells, we performed RT-PCR of RNA samples isolated from human donor retinas and \nhuman retinal organoids using the same primer pairs. We observed a very similar band \npattern compared to the RT-PCR results from skin fibroblasts and PBMCs, suggesting \nno major differences in mRNA splicing of ABCA4 and RPE65 between the different cell \nand tissue types (Fig. 2A). \nAlthough RT-PCR is a robust method for the qualitative detection of mRNA splicing \ndefects, it is less suitable for the reliable quantification o f the results. In addition, \nparticularly for low abundant transcripts, it is susceptible to artificial variations, \nmanifesting during cDNA synthesis and subsequent PCR amplification. To overcome \nthe drawbacks of RT -PCR and to validate our results with addi tional methods, we \nanalyzed RNA from PBMCs of several healthy individuals using two next generation \nRNA sequencing techniques: short -read RNA sequencing (Illumina) and long -read \nRNA sequencing (PacBio Revio, Fig. 3A). Short -read sequencing of samples with \ntranscriptionally activated ABCA4 and RPE65 showed a robust increase in gene \nexpression for both genes in comparison to non -treated control samples (Fig. 3B) . \nSplice junction analysis of ABCA4 demonstrated that th e level of transcriptional  \nactivation achieved with CATALYTEC is in principle sufficient to analyze the different \ntranscript isoforms (Suppl. Fig. 4F).  \nAnalysis of long-read RNA sequencing data in healthy PBMC samples revealed that \nover 30% of detected splice variants exhibit incomplete spli ce matches, i.e. variants \nwith partial alignment but differing at the 5’ or/and 3’-end of the transcript. Additionally, \nmore than 30% of isoforms represent either novel combinations of known splice sites \n(classified as “Novel in catalogue”) or incorporate at least one previously unidentified \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n9 \nsplice site (“Novel not in catalogue”), highlighting the capability of long -read \nsequencing to identify novel splice isoforms (Fig. 3C). \nSimultaneous transactivation of ABCA4 and RPE65 and single transactivation of \nMYO7A led to full transcript coverage by long -read sequencing, corroborating the \nincrease in gene expression as previously determined via RT -qPCR and short -read \nRNA sequencing (Fig. 3D). The respective consensus sequences, constructed by \nclustering similar rea ds and eliminating potential artifacts, were used to identify \nisoforms of biological relevance. For RPE65 and MYO7A, several full-length isoforms \nwere identified as consensus sequences (Suppl. Fig. 6). In contrast, only one \nconsensus sequence was obtained for ABCA4, as multiple transcript reads were \nexcluded in the filtering process. \nFinally, we applied the described CATALYTEC protocol to one patient with confirmed \nbiallelic RPE65-associated LCA (P1), two patients with the clinical phenotype of rod -\ncone dystrophy (RP, P2 and P3) but unclear molecular genetic diagnosis , and three \npatients with the clinical diagnosis of ABCA4-associated Stargardt’s disease (STGD1; \nP4-P6) (Tab. 1, Fig. 4). \nAll patients, except for P 3, had previously undergone molecular genetic testing of \nwhom only P 1 and P4  had a definite molecular genetic diagnosis (compound \nheterozygosity), which was confirmed by segregation analysis. \nP1 is a 6 -year-old male who has been severely visually impaired from birth and \nreceived a genetic diagnosis of RPE65-associated LCA at the age of 5. He was treated \nwith voretigene neparvovec at the age of 6  (15). P2 and P3 are two female siblings \nwho first experienced impaired vision (night blindness and in the further course of the \ndisease visual field restriction) in their early 20’s with the clinical diagnosis of rod-cone \ndystrophy (RP) lacking a definite genetic diagnosis. In 2013, an initial molecular genetic \ntesting via gene panel sequencing in P2 comprising 42 known IRD genes revealed one \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n10 \nheterozygous variant of unknown significance in the RPE65, the CRB1 and the RP1L1 \ngene, respectively (Tab. 1). WES performed in 2021 did not provide any additional \ninformation. \nP4 is a 49-year-old female who first experienced visual deterioration at the age of 33 \nrepresenting a later onset form of STGD with pronounced macular atrophy but foveal \nsparing (Fig. 4). P5 is a 28 -year-old female and P6 is her 31 -year-old brother, both \ndiagnosed with macular dystrophy at the ages of 19 and 21, respectively. \nPotentially pathogenic compound heterozygous mutations in ABCA4 and RPE65 were \nfound in all STGD1 patients (P4-P6) and in the P1 patient, respectively (Tab. 1). \nWe performed the CATALYTEC on PBMCs of all these patients and analyzed the  \nisolated RNA by RT-PCR. P1 carries c.11+5G>A within the splice donor site of exon 1 \nin the RPE65 gene. According to RT -PCR analysis, this mutation does not severely \naffect mRNA splicing at that position (Fig. 5B). For the second mutation affecting the \ncanonical consensus sequence of the splice acceptor site in intron 7  (c.726-2A>T, \nFig.5A), RT-PCR analysis revealed a clear difference in the band pattern compared to \nthe unaffected  control sample , experimentally confirming the splicing divergence . \nSanger sequencing of these bands revealed partial skipping of exon 8 and inclusion of \nintron 7 (Fig. 5B, C). \nThe c.1937+1G>A mutation of P4 disrupts the splice donor site of exon 13 in ABCA4 \n(Fig. 5A). A previous case study has associated this mutation with severe phenotypes, \nincluding STGD and macular degeneration  (16). A recent publication r eported that it \ncauses aberrant splicing by activating cryptic splice donor site s within exon 13 and \nintron 13, ultimately resulting in a 132 bp in-frame deletion (17). This conclusion was \nbased on data obtained with a minigene assay. Potential consequences at the protein \nlevel, such as p.[(Y.603_S646del, F647*)], have not yet been experimentally validated. \nFor this mutation, we visually detected an increased ratio of alternative exon 15 splicing \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n11 \nin comparison to healthy control samples in RT -PCR (Fig. 5B, D). As described in \nTable 1, this alternatively spliced ABCA4 variant leads to an in-frame skipping of exon \n15. However, the significance of this deletion for ABCA4 protein expression, stability \nand function remains unclear and is outside the focus of this study . For all other \npatients carrying potentially pathogenic mutations  in IRD genes analyzed herein , no \nobvious effects on splicing could be detected (Fig. 5B). \nTaken together, these results suggest that our CATALYTEC is in principle suitable for \nthe detection and quantification of splicing mutations in patients’ PMBCs.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n12 \nDiscussion \nHere, we established CATALYTEC, a robust and simple approach for CRISPRa-based \nactivation of genes in human PBMCs and skin fibroblasts suitable for diagnostic \npurposes. \n \nCATALYTEC fulfills various criteria for use in diagnostics: \ni) Due to its simplicity, it is easy to implement in routine diagnostics. \nii) It can be utilized to detect novel mutations in known genes, which might have \nbeen overlooked in the past due to technical limitations. \niii) It can be used to validate the proposed pathogenicity of detected mutations.  \niv) It is without further modifications transferable to other genetic disorders \nwhere biopsy of the affected tissue from the patients is not possible.  \n \nIn recent studies, similar approaches were developed to activate the MPZ and SPAST \ngenes associated with Charcot-Marie-Tooth disease and hereditary spastic paraplegia, \nas well as the CRB1 gene associated with  retinitis pigmentosa,  in human skin \nfibroblasts (18, 19) . Compared to th ese studies, our method offers additional key \nadvancements. We demonstrate for the first time that dCas9 -VPR can effectively \nactivate disease -associated genes in PBMCs  readily isolated from patient blood \nsamples. Skin fibroblasts have the disadvantage that their isolation requires invasive \nskin punching, which is more elaborate and associated with reduced patient \ncompliance. Therefore, genetic testing of human skin fibroblasts is less suitable for \nbroad routine diagnostics compared to PBMCs. Additionally, the isolation and \ncultivation of skin fibroblasts is more time -consuming. With our CATALYTEC \napproach, we achieved a turnaround time from blood collection to first RT-PCR results \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n13 \nof 48 -72h, ultimately increasing the convenience for patients and the healthcare \nsystem. \nWe have applied CATALYTEC to several common genes  associated with  IRDs, a \nheterogeneous group of genetic diseases  affecting the retina . In particular, we have \nproven the ability of our approach to activate large genes  like ABCA4 and MYO7A. \nThis is of great importance as deep intronic mutations in large genes are not covered \nby WES or, when identified by WGS, cannot b e correctly interpreted in terms of their \npotential impact on mRNA splicing. \nOur data shows that the level of transcriptional activation obtained with CATALYTEC \nis high enough  to be combined with the most commonly used readout methods  \n(RT-PCR, RT -qPCR, short - and long -read RNA sequencing). RT -PCR analysis \nprovided the first evidence that CATALYTEC can be used to detect pathogenic splice \nmutations in PBMCs of IRD patients. Additionally, we show that short - and long-read \nsequencing can be  used to analyze the expression and alternative splicing of \ntranscriptionally activated genes in PBMCs. However, a meaningful and significant \nresult for diagnostic purposes necessitates a more detailed analysis of isoforms, which \ncould be achieved through higher coverage of the respective target genes. \nWe demonstrate that the splicing patterns of activated IRD genes in human PBMCs \nand fibroblasts are very similar to those observed in human retinas and retinal \norganoids. Yet, we cannot exclude the possibility that other activated genes in PBMCs \nmight have different splicing patterns than in the cells in which these genes are \nnaturally expressed. In addition, rare tissue -specific splicing events may have been \nmissed by RT -PCR. More detailed investigations using short- and long -read \nsequencing of the human retina and organoid transcriptomes could provide valuable \ninsights in this regard. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n14 \nAn attractive alternative to PBMCs would be an application of the CATALYTEC \nprotocol in buccal epithelial cells, as their collection is technically even simpler and less \ninvasive than blood sampling. It remains to be seen whether this goal can be achieved \nusing CRISPRa or other methods for introducing DNA or RNA into these cells. \nIn summary, we provide proof of concept for a CRISPRa -based approach to activate \nIRD-associated genes in PBMCs in sufficient quantities. The resulting transcripts can \nbe detected  and quantified  using standard methods and  analyzed for structural \nvariants and splicing defects. The CATALYTEC method is universally applicable, can \nbe easily adapted to other target genes, and could help closing important gaps in the \ndiagnosis of inherited (retinal) diseases. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n15 \nMethods \nSex as a biological variable \nOur study examined men and women, as gender should not be an exclusion criterion \ndue to the diagnostic background of the study. \n \nPlasmids \nThe pcDNA3.1-CMV-dCas9-VPR and plasmids for LV  production (pMDL, pRSVRev \nand pMD2.G) were obtained from Addgene  (#63798, #12251, #12253, #12259). \nExpression of dCas9 -VPR in pcDNA3 .1 vectors was driven by the cytomegalovirus \n(CMV) promoter. In lentiviral CRISPRa systems, the dCas9 -VPR expression was \ninduced by either CMV, spleen focus -forming virus (SFFV) or elongation factor 1  \n(EF1) promoter. \nSingle-guide RNA cassettes were synthesized (Azenta , IDT ) and inserted using \nstandard cloning techniques.  Sequences are shown in Suppl. Table 3 . All plasmids \nwere sequenced before use (Eurofins Genomics/Microsynth). \n \nCell Culture \nHEK293T cells (Takara Bio) were maintained in DMEM (high glucose, Thermo Fisher \nScientific) supplemented with 10% FBS ( Superior, Sigma -Aldrich) and 1% \npenicillin/streptomycin (P/S, Thermo Fisher Scientific) at 37 °C, 10 % CO2. \nHuman skin fibroblasts ( adult, Sigma-Aldrich) were cultured in DMEM (low glucose) \nsupplemented with 10% FBS and 1% P/S at 37 °C, 5% CO2. \nPBMCs were cultured in RPMI -1640 ( Thermo Fisher Scientific) supplemented with \n10% FBS at 37 °C, 5% CO2. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n16 \nCollection and stimulation of PBMCs \nPBMCs were isolated through density centrifugation of the collected whole blood. The \nisolated buffy coat was washed with PBS and  resuspended in RPMI -1640, \nsupplemented with 10% FBS and 1X phytohemagluttinin-L ( PHA-L, Thermo Fisher \nScientific). The cells were incubated at 37 °C and 5% CO2 for 20 h before being used \nfor nucleofection. \n \nIsolation of human retinas \nHuman donor eyes without cornea and lens were received from the eye  bank of the \nUniversity Hospital Zurich in ice -cold PBS. Retina and eyecups were separately flat \nmounted after making four incisions. Retina samples were collected from the nasal \nperiphery and the macular region. The isolated retina  was snap-frozen and stored at  \n-80 °C until further processing. \n \nTransfection of cell lines and primary cells \nHEK293T were transfected with  either XfectTM (Takara Bio) or Lipofectamine 3000 \n(Invitrogen) according to the manufacturers’ instructions and harvested 48  h after \ntransfection. \nPHA-L-stimulated PBMCs (2.0-2.5x10^7 cells per reaction) were nucleofected with the \nP3 Primary Cell 4D -Nucleofector Kit  (Lonza). After application of program EO -115, \n500 µL of pre-equilibrated culture medium was added to the cuvette and the  \nsuspension was immediately transferred to a pre-equilibrated 12-well plate . \nNucleofected PBMCs were incubated for 24 h at 37 °C, 5% CO2. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n17 \nFor nucleofection of  fibroblasts (2x10^6 cells per reaction) the P2 Primary Cell 4D -\nNucleofector Kit (Lonza) was used. After application of program CZ-167, 500 µL of pre-\nequilibrated culture medium was added to the cuvette and the suspension was \nimmediately transferred to a pre -equilibrated 6 -well plate . N ucleofected fibroblasts \nwere incubated for 24 h at 37 °C, 5% CO2. \n \nRNA isolation \nHEK293T and fibroblasts were washed with PBS and lysed with RLT plus buffer \n(Qiagen) supplemented with 10 µL/mL -mercaptoethanol (Carl Roth). The cell lysate \nwas transferred into safe-lock tubes (Eppendorf) and homogenized with a mixer mill \n(Retsch) at 30 Hz for 1 min. After centrifugation, RNA was isolated according to the \nmanufacturer’s protocol of the RNeasy Plus Mini Kit (Qiagen). \nFor PBMCs, the cell were pelleted and lysed with RLT plus buffer supplemented with \n10 L/mL -mercaptoethanol. Subsequently, the lysate was added to  QIAshredder \nhomogenization colum ns (Qiagen) and centrifuged according to the manufacturers’ \nprotocol. After homogenization, the RNeasy plus Mini Kit was used to isolate the RNA. \nSnap-frozen retinas were lysed in buffer RLT supplemented with 10 µL/mL  \n-mercaptoethanol. The retinas were homogenized through a 21G needle. Afterwards, \nthe RNA was isolated with the RNeasy Plus Mini Kit. \nRNA isolation from human retinal organoids was done with the Direct -zol DNA/RNA \nminiprep kit (Zymo) according to the manufacturer's instructions. \nTo avoid gDNA contamination, an on-column DNase I digest (Qiagen) was performed \nfor every RNA isolation. \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n18 \nTwo-step RT-PCR \nFirst-strand cDNA for two-step RT-PCR analysis of HEK293T, PBMCs and fibroblasts \nwas produced with Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific) \naccording to the manufacturers’ protocol.  First-strand cDNA from retina l RNA was \nsynthesized with the M-MLV reverse transcriptase (Promega). \nSubsequent second-strand synthesis and PCR amplification was performed with the \nQ5 Hot Start High Fidelity Polymerase ( New England Biolabs ). Primer s used for \namplification are listed in Suppl. Tab le 4. Results were visually analyzed through \nagarose gel electrophoresis (1% w/v). Amplified bands were isolated with the QIAquick \nGel Extraction Kit (Qiagen) and sent for Sanger sequencing (Microsynth AG, Eurofins). \n \nRT-qPCR \nFor quantitative real -timer PCR, RNA was reverse transcribed into cDNA with the \nRevertAid First Strand cDNA synthesis Kit (Thermo  Fisher Scientific). The SYBRTM \nGreen PCR Master Mix (Thermo  Fisher Scientific) was used to prepare the samples \naccording to th e manufacturers’ instructions. For amplification and analysis, the \nMicroAmpTM Fast Optical 96-Well Reaction Plate and QuantStudio 3 RT-PCR system \nand software (Thermo Fisher Scientific) were used. Expression levels were normalized \nto ALAS1. Primers used for RT-qPCR analysis are listed in Suppl. Table 5. \n \nOptical coherence tomography (OCT) and autofluorescence (FAF) imaging \nRetinal cross sections were obtained with spectral -domain optical coherence (SD -\nOCT) tomography using the Heidelberg Spectralis  OCT (Heidelberg Engineering) . \nFAF (488 nm) was obtained using the same device.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n19 \nLentivirus production \nFor production of lentiviral vectors, HEK293T were transfected with pMDL, pRSVRev, \npMD2.G as well as the transgene plasmid plasmids via calcium phosphate method. \nThe transfected cells were incubated for 48 h at 37 °C, 10% CO2. Subsequently, the \ncell culture medium was collected and filtered with a 0.45 µm filter unit (VWR). The \ncells were supplied with fresh culture medium and incubated for additional 24 h. The \nfiltered medium was centrifuged at 19400 rpm and 17  °C for 2 h (Beckman Coulter). \nThe pellet was suspended in 250 µL HBSS (Thermo Fisher Scientific ) and stored at \n4 °C overnight. The described procedure was repeated with the cell culture media, \nadded the day before. The first and second harvest were combined and concentrated \nwith a sucrose cushion centrifugation at 21000 rpm, 17 °C for 2 h. The resulting pellet \nwas suspended in 7 0 µL HBSS an d mixed for 45 min at 1400 rpm . Final aliquots of \n5 µL were stored at -80 °C. \nTiter determination was performed with a RT-qPCR Lentivirus Titer Kit (Applied \nBiological Materials Inc.) according to the manufacturers’ instructions. \n \nGeneration of human retinal organoids \nHuman retinal organoids were differentiated from the human derived iPSCs (F49B7). \nPluripotency markers and germ layer differentiation potential was determined as \npreviously described  (20). I PSCs were  seeded in matrigel -coated 6 -well plates \n(Corning) and cultured in mTeSR plus medium (STEMCELL) at 37  °C, 5% CO 2. The \niPSCs were passaged using 0.5 mM EDTA (pH 8.0, Thermo Fisher Scientific). \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n20 \nDifferentiation of iPSCs into human retinal organoids was performed according to the \nprotocol developed by Kim et al  (21), with some modifications. All organoids at the \ndifferent maturation stages were cultured in a humidified incubator at 37 °C, 5% CO2. \nOn day 0, the iPSCs were dissociated using 0.5 mM EDTA. The aggregates were \nsuspended in cold Matrigel (GFR, Corning) and incubated at 37  °C for 20 min. \nAfterwards, the iPSC/Matrigel aggregates were dispersed in neural induction medium \n(DMEM/F12 with neurobasal medium (1:1) supplemented with 1% B27 (incl. vitamin A \nsupplement), 0.5% N-2 supplement, 0.1 mM -mercaptoethanol, 2 mM GlutaMax and \n1% P/S (all Thermo Fisher Scientific)) and cultivated in ultra-low adherent 6-well plates \n(Costar®, Corning). On day 5, floating cysts were seeded in matrigel -coated 6-well \nplates. On day 15, cysts were detached by adding dispase (0.5 mg/mL in DMEM/F12, \nSTEMCELL), washed with DMEM/F12 and further cultured in retin al differenti ation \nmedium (DMEM/GlutaMax supplemented with F12 nutrition mix (3:1), 2% B27 (without \nvitamin A), 1% non -essential amino acids (NEAA) and 1% P/S). On day 25, the \nimmature retinal organoids were transferred to  retinal maturation medium \n(DMEM/GlutaMax suppl emented with F12 nutrition mix (3:1), 8% FBS, 2% B27 \n(without vitamin A), 1% NEAA,  1% antibiotic/antimycotic  and 1% 100 mM taurine \n(Sigma-Aldrich). \nOn day 230, mature organoids were used for RNA isolation, AAV transduction and \nsubsequent experiments. \n \nShort-read RNA sequencing of healthy blood samples \nFor short read mRNA sequencing, 100 ng of DNase treated total RNA (RIN > 8) was \nprocessed with the TruSeq Stranded mRNA Prep Kit (Illumina) including poly(A) \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n21 \nselection. Indexes were added with the Illumina  RNA UDI 384 v2 kit (IDT). 150 bp \npaired end sequencing was performed with a NovaSeq X system  (Illumina). \nSequenced reads were aligned to the human reference genome (GRCh38) and were \ncounted on gene -level with Rsubread (version 2.18 ).  \nDifferentially expressed genes (DEGs) were determined between  \ncontrol and treated samples employing edgeR (v4.2.1) and limma (v3.60.3). Gene set \nenrichment analysis of DEGs was carried out with fgsea (v1.30.0). Sashimi blots were \ngenerated with Integrative Genomics Viewer (IGV, version 2.17.4 03/26/2024). \n \nLong read RNA sequencing of healthy blood samples \nFor PacBio long -read sequencing healthy blood samples were sent to an external \nsequencing facility (Bioscientia). Sequencing was performed on a PacBio Revio device \n(Pacific Biosciences). The Isoseq (v4.0.0; https://isoseq.how/) workflow was utilized to \nanalyze Hifi reads and quantify gene expression, as well as, extract different transcript \nisoforms. Mapped long reads were visualized in IGV. \n \nWES of patient-derived blood samples \nGenomic DNA, isolated from collected blood samples, was fragmented, and the coding \nexons of the analyzed genes as well as the corresponding exon -intron boundaries \nwere enriched using Roche/KAPA sequence capture technology (KAPA HyperExome \nLibrary) and sequenced using an Illumina NovaSeq 6000 system. The requested gene \npanel was extracted from the WES data. The target regions were sequenced with an \naverage coverage of 337x. For more than 99% of the target regions a 15-fold coverage \nwas obtained. Putatively pathogenic differences between the wildtype sequence \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n22 \n(human reference genome according to UCSC Genome Browser: hg19, GRCh37) and \nthe interpreted patient's sequence were assessed using an internally established \nquality system. Variants, which did not pass the quality threshold, were verified using \nconventional Sanger sequencing. Variants listed as additional, putatively relevant \nvariants were not routinely validated. Identified variants were compared to literature \nand external as well as internal allele frequency databases (e.g. gnomAD). In addition, \nin silico  analysis of the identified variants with regard to functional relevance, \nconservation and splice effects was performed using bioinformatic prediction programs \n(e.g. SpliceA I, MaxEntScan). The variants were classified using the current ACMG \nguidelines (22). \n \nLong-read WGS of patient-derived blood samples \nGenomic DNA, isolated from collected blood samples, was fragmented, and a PCR -\nfree library was prepared using SMRTbell prep kit 3.0  (PacBio). Long-read whole -\ngenome sequencing was done using a PacBio Revio system at an average coverage \nof approx. 30-fold. Putatively pathogenic differences between t he wildtype sequence \n(human reference genome according to UCSC Genome Browser: hg19, GRCh37) and \nthe patient's sequence mentioned and interpreted in this report were assessed using \nan internally established quality system.  \nIdentified variants were compare d to literature and external as well as internal allele \nfrequency databases  (e.g. gnomAD) . In addition, in silico  analysis of the identified \nvariants with regard to functional relevance, conservation and splice effects was \nperformed using bioinformatic pre diction programs. The variants are classified using \nthe current ACMG guidelines (22). \nThe genome data we re filtered with respect to autosomal recessive, autosomal \ndominant and X -linked mode of inheritance for very rare potentially pathogenic \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n23 \nhomozygous/putative compound heterozygous/heterozygous and hemizygous \nchanges. For the filtered variants, a literature-based comparison using human mutation \ndatabases (e.g. HGMD, ClinVar) was performed according to the provided clinical \ninformation of the patient. In addition, the data were compared to public and internal \nallele frequency databases. Furthermore, the in-silico scores of bioinformatic prediction \nprograms were also taken into account. The NGS data were not analyzed for \npotentially pathogenic variants in genes not related to the requested indication. \nAdditional, putatively relevant variants and carriership findings are n ot reported \nroutinely. \n \nStatistical analysis and reproducibility \nAll values are given as mean ± SEM. Statistics were performed with GraphPad PRISM \n(GraphPad Software, v10.0.2). \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n24 \nStudy approval \nThe patients (P1 -P6) involved in this project presented at the Department of \nOphthalmology at LMU Munich and research including patient samples were approved \nby the local ethics committee (ethics vote nr. 19 -0226). Clinical research and \npublication of clinical imaging data was approved by the local ethics committee (ethics \nvote nr. 22-0897). Written informed consent for the use of patient samples and clinical \nimaging was received prior to participation and has been retained. \nHuman eyes, used for human retin a analysis, were donated and collected in \ncollaboration with the Eye Clinic Zurich and were approved by the local ethics \ncommittee (BASEC-Nr: PB_2017-00550 and 2020-01856). \nAll procedures with human samples and donations adhered to the tenets of the \nDeclaration of Helsinki. \n \nData availability \nValues for all data points shown in graphs are reported in the Supporting Data Value \nfile. \nSequencing data of human samples have not been deposited  in a public repository \nbecause this could compromise the privacy of the research participants, but they are \navailable from the corresponding author upon request. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n25 \nAuthor contributions \nV.J.W., K.S.H., S.M. and E.B. designed the research studies. V.J.W., M .J.G., A.R., \nK.S.H., H.J.B. and D.Y.O. conducted the experiments and acquired data. T.H., V.J.W. \nanalyzed data. F.B. and I.M. provided human retina samples. V.J.W., M.J.G and E.B. \nwrote the manuscript.  E.B., S.M. and M.B.  acquired funding. S.M. and M.J.G. \nsupervised experiments with human patient samples. E.B. supervised the project. All \nauthors contributed to the final manuscript. \n \nAcknowledgements \nThis research study was funded by the Helmut -Ecker Stiftung  (to E.B.) , Novartis \nStiftung (to S.M.) , Iten -Kohaut Stiftung  (to E.B.) and the Swiss National Science \nFoundation (to E.B., 320030E_221942). \nWe thank Bioscientia Healthcare GmbH for performing the library preparation and the \nsequencing for the PacBio long-read RNA sequencing approaches. \nWe thank Claudia Matter for the processing of human eye donations. \nLibrary preparation and sequencing for short-read RNA sequencing was performed by \nthe Functional Genomics Center Zurich (FGCZ). \nFigure 3A and the graphical abstract  were created with BioRender.com \n(https://app.biorender.com/).  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n26 \nCompeting financial interests \nE.B., S.M. and M.B.  are authors on a patent application covering trans criptional \nactivation of (retinal) genes for diagnostic purposes (PCT/EP2020/076536, filed by \nViGeneron GmbH). S.M. and M.B. are co-founders and shareholders of ViGeneron \nGmbH and members of its scientific advisory board. E.B. is a member of the scientific \nadvisory board of ViGeneron GmbH. The remaining authors declare no competing \ninterests.  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n27 \nReferences \n1. Graessner H, et al. Solving the unsolved rare diseases in Europe. Eur J Hum Genet. \n2021;29(9):1319-1320. doi:10.1038/s41431-021-00924-8 \n2. van El CG, et al. Whole-genome sequencing in health care: recommendations of the \nEuropean Society of Human Genetics. Eur J Hum Genet. 2013;21(6):580-584. \ndoi:10.1038/ejhg.2013.46 \n3. Chrystoja CC, Diamandis EP. Whole genome sequencing as a diagnostic test: challenges \nand opportunities. Clinical chemistry. 2014;60(5):724-733. \ndoi:10.1373/clinchem.2013.209213 \n4. Maggi J, et al. Nanopore Deep Sequencing as a Tool to Characterize and Quantify \nAberrant Splicing Caused by Variants in Inherited Retinal Dystrophy Genes. 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RNA. 2018;9(1). doi:10.1002/wrna.1451 \n10. Lynn N, Tuller T. Detecting and understanding meaningful cancerous mutations based on \ncomputational models of mRNA splicing. NPJ systems biology and applications. \n2024;10(1):25. doi:10.1038/s41540-024-00351-7 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n28 \n11. Chavez A, et al. Highly efficient Cas9-mediated transcriptional programming. Nature \nmethods. 2015;12(4):326-328. doi:10.1038/nmeth.3312 \n12. Chavez A, et al. Comparison of Cas9 activators in multiple species. Nat Methods. \n2016;13(7):563-567. doi:10.1038/nmeth.3871 \n13. Frimpong K, Spector SA. Cotransduction of nondividing cells using lentiviral vectors. \nGene therapy. 2000;7(18):1562-1569. doi:10.1038/sj.gt.3301283 \n14. Vora S, et al. Rational design of a compact CRISPR-Cas9 activator for AAV-mediated \ndelivery. bioRxiv. 2018:298620. doi:10.1101/298620 \n15. Gerhardt MJ, et al. Gene Therapy with Voretigene Neparvovec Improves Vision and \nPartially Restores Electrophysiological Function in Pre-School Children with Leber \nCongenital Amaurosis. Biomedicines. 2022;11(1). doi:10.3390/biomedicines11010103 \n16. Eandi CM, et al. Homozygous c.1937+1GA splice-site variant of the ABCA4 gene is \nassociated with Stargardt disease. European journal of ophthalmology. 2014;24(5):814-\n817. doi:10.5301/ejo.5000458 \n17. Schulz HL, et al. Mutation Spectrum of the ABCA4 Gene in 335 Stargardt Disease \nPatients From a Multicenter German Cohort-Impact of Selected Deep Intronic Variants \nand Common SNPs. Invest Ophthalmol Vis Sci. 2017;58(1):394-403. doi:10.1167/iovs.16-\n19936 \n18. Terkelsen T, et al. CRISPR activation to characterize splice-altering variants in easily \naccessible cells. American journal of human genetics. 2024;111(2):309-322. \ndoi:10.1016/j.ajhg.2023.12.024 \n19. Moon SY, et al. Rapid Variant Pathogenicity Analysis by CRISPR Activation of CRB1 \nGene Expression in Patient-Derived Fibroblasts. CRISPR J. 2024;7(2):100-110. \ndoi:10.1089/crispr.2023.0065 \n20. Cowan CS, et al. Cell Types of the Human Retina and Its Organoids at Single-Cell \nResolution. Cell. 2020;182(6):1623-1640.e34. doi:10.1016/j.cell.2020.08.013 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n29 \n21. Kim S, et al. Generation, transcriptome profiling, and functional validation of cone-rich \nhuman retinal organoids. Proceedings of the National Academy of Sciences of the United \nStates of America. 2019;116(22):10824-10833. doi:10.1073/pnas.1901572116 \n22. Richards S, et al. Standards and guidelines for the interpretation of sequence variants: a \njoint consensus recommendation of the American College of Medical Genetics and \nGenomics and the Association for Molecular Pathology. Genetics in medicine : official \njournal of the American College of Medical Genetics. 2015;17(5):405-424. \ndoi:10.1038/gim.2015.30 \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n30 \nGraphical abstract \n \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n31 \n \nFigure 1 Transcriptional activation of IRD -associated genes in transfected \nHEK293T. A, Scheme depicting the expression plasmid and the corresponding dCas9-\nVPR protein bound to a gene of interest (GOI). dCas9 -VPR, driven by either CMV or \nSFFV promoter, was combined with a cassette expressing sgRNAs, targeting either \nRPE65, ABCA4, MYO7A or USH2A. For simultaneous gene activation multiple sgRNA \ncassettes were combined. pA, poly A signal. B, Primer design for RT -PCR analysis. \nPrimer binding sites (black arrows) and PCR product lengths in base pairs (bp) are \nshown in the schematic representation of the transcripts. Colored boxes represent \nnumbered exons of the respective genes. UTR, untranslated region. C, RT-PCR result \nfor each gene transcript after transcriptional activation in HEK293T cells. kbp, kilo base \npairs. D, RT-qPCR result for each gene  activation. MX, multiplexed gene \ntranscriptional activation by combination of multiple sgRNA cassettes. R65, RPE65; \nM7, MYO7A; A4, ABCA4; U2A, USH2A. Values are shown as mean ± SEM. Statistics \nwere calculated with Student’s t-test. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n32 \n \nFigure 2 RT-(q)PCR analyses of transcriptionally activated genes in human PBMCs and \nfibroblasts. A, RT-PCR results for RPE65 (left) and ABCA4 (right). Transcriptionally activated \nPBMCs and fibroblasts are compared to endogenous ly expressed RPE65 and ABCA4 from \nhuman retinal organoids (hROs) and human retina (hRE). B, RT-qPCR results for \ntranscriptionally activated RPE65 (R65) and ABCA4 (A4) in PBMCs (left) and  fibroblasts \n(right). MX, multiplexed transcriptional activation of both genes. Values are given a s mean ± \nSEM. Statistics are calculated with Student’s t-test. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n33 \n \nFigure 3 Short- and long-read RNA sequencing of transcriptionally activated PBMCs. A, \nWorkflow diagram from PBMC isolation to short - and long -read RNA sequencing . B, \nNormalized fragment counts for RPE65 (left, blue) and ABCA4 (right, orange) of four analyzed \nsamples (#1-4) compared to untreated control samples. CPM, counts per million. C, Proportion \nof different structural variant categories after long -read sequencing, clustering and \nclassification. Total number represents the value of 100% for each sample. D, Total transcripts \ncovering reads for either ABCA4 (orange), RPE65 (blue) or MYO7A (green) after long-read \nsequencing. Dark blue schemes below represent the reference transcripts of the respective \ngenes. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n34 \n \nFigure 4 Clinical phenotype of patients. A, Top panel shows optical coherence tomography \n(OCT), 30° and 55° fundus autofluorescence (AF) images of a healthy subject (control). Three \nlower panels show  the corresponding images of three individuals (P1 -P3) with confirmed \nRPE65-associated retinal disease (RPE65-LCA and RP). P1 shows no autofluorescence due \nto severe RPE65 deficiency. B, OCT and AF images of one healthy subject (control) and three \nindividuals (P4 -P6) with confirmed ABCA4-associated retinal disease  (STGD1). AF, \nautofluorescence. OD, oculus dexter. OS, oculus sinister. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n35 \n \nFigure 5  RPE65 and ABCA4 transcript analysis in patient samples.  A, Scheme \nhighlighting the position of the c.726-2A>T mutation in RPE65 (patient P1 ; blue) and \nc.1937+1G>A mutation in ABCA4 (patient P4; orange). Colored boxes represent exons, the \nline in between represents the intron. B, RT-PCR results for patients P1 – P6 in comparison \nto a healthy human control sample (CTRL). C, Scheme summarizing Sanger sequencing result \nof the numbered bands 1 and 2 of sample P1 shown in B. PTC, premature termination codon. \nD, Scheme summarizing the results of RT -PCR analysis of the num bered bands 1 – 3 from \nsample P4, as indicated in B. \n  \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint \n\n \n36 \nTable 1: Genetic diagnosis for each patient and sequencing methods \nPatient Gene and mutation Comments \nP1 RPE65, c.11+5G>A, heterozygous; \nRPE65, c.726-2A>T, heterozygous \nExome sequencing A (Twist Hu man \nCore Exome + Twist Human RefSeq \npanel); Compound heterozygosity \nconfirmed by segregation analysisB \nP2 RPE65, c.245+5A>G, heterozygous; \nCRB1, c.585C>G, heterozygous; \nRP1L1, c.2465G>A, heterozygous \nGene panel diagnostics comprising 42 \nknown IRD genes; WES did not reveal \nadditional information; Sibling of P3 \nP3 No genetic testing result available Clinical phenotype of RP; Sibling of P2 \nP4 ABCA4, c.1937+1G>A, heterozygous; \nABCA4, c.5603A>T, heterozygous; \nABCA4, c.1009T>C, heterozygous \nDirect sequencing of the ABCA4 gene \nand segregation analysisC \nP5 ABCA4, c.2966T>C, heterozygous; \nABCA4, c.5603A>T, homozygous \nWES; Sibling of P6 \nP6 ABCA4, c.2966T>C, heterozygous; \nABCA4, c.5603A>T, homozygous \nWES; Sibling of P5 \nATwist Human Core Exome combined with Twist Human RefSeq panel; Bc.11+5G>A \nwas detected in the mother and c.726-2A>T was detected in the father of P1; CMother \nof P4 is carrier of the heterozygous missense mutations c.5603A>T and c.1009T>C. \nThe father of P4 was not available for segregation analysis. \n \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted December 1, 2024. ; https://doi.org/10.1101/2024.11.29.625601doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}