# Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in presenilin 1 gene

Coralie Clua Provost, Louise Greetham, Cécile Monzo, Arnaud Monteil, Anne Rovelet-Lecrux, Sylvain Lehmann, David Wallon, Valentin Garcia, Hélène Hirbec, Emmanuel Nivet, et al.

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Coralie Clua Provost, Louise Greetham, Cécile Monzo, Arnaud Monteil, Anne Rovelet-Lecrux, et al. Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in presenilin 1 gene. Stem Cell Research, 2025, 87, pp.103780. 10.1016/j.scr.2025.103780. hal-05213438

HAL Id: hal-05213438

https://hal.science/hal-05213438v1

Submitted on 18 Aug 2025

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Lab Resource: Genetically-Modified Multiple Cell Lines

Generation of two isogenic-corrected control cell lines (IRMBi001-A-1; IRMBi001-A-2) from Autosomal dominant Alzheimer's disease patient-derived iPSCs carrying a G217D mutation in *presenilin 1* gene

Coralie Clua Provost<sup>a</sup>, Louise Greetham<sup>b</sup>, Cécile Monzo<sup>a</sup>, Arnaud Monteil<sup>c,d</sup>,

Anne Rovelet-Lecrux<sup>e</sup>, Sylvain Lehmann<sup>a</sup>, David Wallon<sup>f</sup>, Valentin Garcia<sup>c</sup>, Hélène Hirbec<sup>c</sup>,

Emmanuel Nivet<sup>b</sup>, Carole Crozet<sup>a,†</sup>

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A B S T R A C T

Mutations in the *presenilin1* (PSEN1) gene are responsible for rare autosomic dominant Alzheimer's disease (ADAD). We generated isogenic control cell lines from iPS cell line derived from ADAD patient carrying a G217D mutation in PSEN1 gene, with CRISPR Cas9 technology. The edited cell lines present the correction of the c.650G > A mutation, no chromosomal abnormalities and no evidence of off-target event. The IRMBi001-A-1 and IRMBi001-A-2 cell lines exhibit pluripotency markers expression and the ability to differentiate into the three germ layers. These two isogenic controls will be used as control to study the pathomechanistic of ADAD through various *in vitro* assays.

Resource Table

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi001-A-1 (INMi006-1); IRMBi001-A-2 (INMi006-2)</td></tr><tr><td>Alternative name(s) of stem cell line</td><td>AD-PS1-ISO1; AD-PS1-ISO2</td></tr><tr><td>Institution</td><td>Institute for Neurosciences of Montpellier</td></tr><tr><td>Contact information of the reported cell line distributor</td><td>Carole Crozet: carole.crozet@inserm.fr</td></tr><tr><td>Type of cell line</td><td>iPSC</td></tr><tr><td>Origin</td><td>Human</td></tr><tr><td>Additional origin info</td><td>Age: 50 y.o.</td></tr><tr><td>(applicable for human ESC or iPSC)</td><td>Sex: Male</td></tr><tr><td>Cell Source</td><td>Fibroblasts</td></tr><tr><td>Method of reprogramming</td><td>Sendai virus cytotune kit</td></tr><tr><td>Clonality</td><td>Clonal</td></tr><tr><td>Evidence of the reprogramming transgene loss (including genomic copy if applicable)</td><td>Clonal isolation approach: Clones were manually picked<br>RT-/q-PCR</td></tr></table>

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Resource Table (continued)

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi001-A-1 (INMi006-1); IRMBi001-A-2 (INMi006-2)</td></tr><tr><td>The cell culture system used</td><td>After reprogramming, iPS cells were grown on MEFs for several passages before adaptation on feeder-free culture using geltrex-coated dishes in mTeSR Plus medium.</td></tr><tr><td>Type of the Genetic Modification</td><td>Mutation Correction</td></tr><tr><td>Associated disease</td><td>Alzheimer's disease</td></tr><tr><td>Gene/locus modified in the reported transgenic line</td><td>Gene: PSEN1<br>Location: exon 7, chromosome 14<br>Mutation: c.650G > A p.Gly217Asp, heterozygous mutation</td></tr><tr><td>Method of modification / user-customisable nuclease (UCN) used, the resource used for design optimisation</td><td>Method of modification: CRISPR/Cas9<br>Design optimisation: CRISPOR</td></tr><tr><td>User-customisable nuclease (UCN) delivery method</td><td>Encoding plasmid transfection and ssODN delivery by electroporation</td></tr></table>

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<footnote><sup>a</sup> Institute for Neurosciences of Montpellier, Univ Montpellier, INSERM U1298, Montpellier, France</footnote>

<footnote><sup>b</sup> Aix-Marseille Univ, CNRS, INP, Inst of Neurophysiopathol, Marseille, France</footnote>

<footnote><sup>c</sup> Institute for Functional Genomics (IGF), Univ Montpellier, CNRS, INSERM, Montpellier, France</footnote>

<footnote><sup>d</sup> Department of Physiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand</footnote>

<footnote><sup>e</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Genetics and CNR-MAJ, Normandie Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote><sup>f</sup> Normandie Univ, UNIROUEN, INSERM U1245, Rouen University Hospital, Department of Neurology and CNR-MAJ, Normandie Center for Genomic and Personalized Medicine, F 76000 Rouen, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: carole.crozet@inserm.fr (C. Crozet).</footnote>
**Resource Table (continued)**

<table><tr><td>Unique stem cell line identifier</td><td>IRMBi001-A-1 (INMi006-1); IRMBi001-A-2 (INMi006-2)</td></tr><tr><td>All double-stranded DNA genetic material molecules introduced into the cells</td><td>Cas9 plasmids: pSpCas9(BB)-2A-Puro PX459 plasmid</td></tr><tr><td>Analysis of the nuclease-targeted allele status</td><td>HDR donor template: ssODN Sanger sequencing PCR products</td></tr><tr><td>Homozygous allele status validation</td><td>Homozygous for wild-type codon 650 of PSEN1 gene (Sanger sequencing + ddPCR)</td></tr><tr><td>Method of the off-target nuclease activity prediction and surveillance</td><td>Targeted top 6 predicted off-target site PCR/sequencing (CRISPOR prediction)</td></tr><tr><td>Descriptive name of the transgene</td><td>N/A</td></tr><tr><td>Eukaryotic selective agent resistance cassettes (including inducible, gene/cell type-specific)</td><td>Puromycin resistance (positive selection)</td></tr><tr><td>Inducible/constitutive expression system details</td><td>N/A</td></tr><tr><td>Date archived/stock creation date</td><td>01/29/2025</td></tr><tr><td>Cell line repository/bank</td><td>Cell lines were deposited on Human Pluripotent Stem Cell Registry https://hpscreg.eu/cell-line/IRM Bi001-A-1 https://hpscreg.eu/cell-line/IRM Bi001-A-2</td></tr><tr><td>Ethical/GMO work approvals</td><td>DC-2015-2363 regional scientific ethical committee CPP Sud Med IV informed consent was obtained from patient. Approval for the use of GMO is active (DUO n° 10982 C2d).</td></tr><tr><td>Addgene/public access repository recombinant DNA sources' disclaimers (if applicable)</td><td>pSpCas9(BB)-2A-Puro (PX459) V2.0 was a gift from Feng Zhang (Addgene plasmid #62988; https://n2t.net/addgene:62988; RRID: Addgene_62988)</td></tr></table>

**1. Resource utility**

Several specific mutations in *PSEN1* are known to be associated to autosomic dominant forms of Alzheimer's disease. This isogenic control cell line will be a powerful tool to study ADAD *in vitro* and allow a fair comparison between healthy and disease phenotypes.

**2. Resource Details**

Our team has previously generated an iPS cell line from a patient carrying the G217D mutation in *PSEN1* gene (IRMBi001-A), associated with autosomic dominant Alzheimer's disease (Auboyer et al. 2019). Here, we reported the generation of two corresponding isogenic control clones through CRISPR/Cas9 technology. Single-guide RNA (sgRNA), able to specifically bind an intra-exonic NGG PAM region of *PSEN1*, near the targeted 650 G > A mutation, was cloned into the pSpCas9(BB)-2A-Puro V2.0 plasmid, coding for the spCas9 gene to induce double-stranded breaks. The plasmid was co-nucleofected with a 127 base-pair long single-stranded oligodeoxynucleotides (ssODN), designed to contain locus-specific homology arms, and the corrected mutation (Fig. 1. panel A), to generate a homologous-directed repair (HDR). To prevent the Cas9 to cut again into the repaired genome, a silent mutation was also inserted into the PAM site of the ssODN. The plasmid also carried a puromycin resistance cassette for the selection of successfully transfected clones. The absence of plasmid integration was confirmed by PCR targeting the plasmid puromycin resistance gene (Supplementary Data S3). A pre-screen was performed based on BstYI digestion profile (unique restriction site only with 650 G > A mutation) (Supplementary Data S1). Sanger sequencing was performed to confirm the successful mutation correction (Fig. 1. panel B) and the absence of unintended allelic dropout at the locus was evaluated by droplet digital PCR (ddPCR) (Supplementary Data S6). The two selected clones presented a normal karyotype with no recurrent genomic abnormalities (Fig. 1.

panel D, Supplementary Data S4) and were mycoplasma free (Supplementary Data S2). Gene-edited clones exhibited typical iPSC morphology (Fig. 1. panel C) and expressed pluripotency markers, OCT4, SOX2 and NANOG (Fig. 1. panel E). The capacity of cells to differentiate into the 3 germ layers was assessed with a trilineage differentiation kit and immunofluorescence of specific lineage markers: PAX6/TUJ1 for ectoderm, BRACHYURY/αSMA for mesoderm and SOX17/FOXA2 for endoderm (Fig. 1. panel F). The absence of DNA alterations on the top 6 predicted off target sites was also confirmed by sequencing (Supplementary Data S5). The isogenic controls shared the same STR profile with the parental cell line (Supplementary files).

**3. Materials and Methods**

**3.1. Induced pluripotent stem cell culture**

iPS cells were cultured on 0.1 % geltrex-coated dishes in mTeSR Plus medium (Stem Cell Technologies) supplemented with 1 % penicillin/streptomycin (Gibco), in a humidified incubator at 37 °C, 5 % CO2. iPS cells were passaged twice a week as aggregates with ReLeSR reagent (Stem Cell Technologies).

**3.2. CRISPR/Cas9 genome editing**

The bioinformatics tool CRISPOR (crispor.tefor.net) was used to design a sgRNA. Forward and reverse primers (Integrated DNA technologies, Table 2) were annealed and ligated into a modified version of the vector pSpCas9(BB)-2A-Puro V2.0 (PX459, Addgene) as previously described (Arnaud et al., 2022; Ran et al., 2013). For genome editing, iPS cells (IRMBi001-A) were prepared and electroporated as previously described in detail (Arnaud et al. 2022). Briefly, plasmid with sgRNA and ssODN were co-nucleofected (Neon™ Transfection System, Invitrogen) into iPS cells. Following puromycin selection, individual transfected clones were manually picked and tested for correction of the targeted mutation by PCR analysis. PCR products were digested with BstYI (3 h at 60 °C) and analysed on a 3 % agarose gel. The expected profiles were as follows: non-corrected (G217D mutation) clones: 149 and 152 bp amplicons in addition to the 301 bp amplicons on the non-mutated allele; corrected clones: 301 bp amplicons (absence of mutation on both alleles). After amplification, positive clones were validated by Sanger Sequencing (described below). Top 6 predicted off target sites (CRISPOR) were analysed by Sanger sequencing. The non integration of the plasmid was also confirmed by PCR as described below.

**3.3. Sanger sequencing and allele copy number assay**

Genomic DNA was extracted from cell pellet with Easy-DNA™ kit (Thermo Fisher Scientific). The targeted region was amplified by PCR with AmpliTaq Gold™ 360 Master Mix (Thermo Fisher Scientific) and analysed with BigDye Terminator v3.1 Cycle Sequencing kit (Thermo Fisher Scientific) on SeqStudio Genetic Analyser. Chromatograms and FASTA sequences were analysed respectively with Chromas (Technelium Pty Ltd) and YOST tool (http://yost.genetics.utah.edu/PoyPeakParser/).

Allele copy number assay was assessed by droplet digital PCR (ddPCR) using supermix for Probes (No dUTP) (Bio-Rad), following the manufacturer's recommendations. The dPCR probe for PSEN1 employed to verify the absence of unintended genomic deletions in the PSEN1 gene, covers a part of the ssODN and is precisely described in Table 2 and Supplementary Data S6. The dPCR probe used for the reference gene SF3B1 (on Chromosome 2) was designed by Qiagen to target a region distant from any gRNA sites used in the gene editing experiment and serves as a stable reference gene for copy number normalization. The PSEN1 probe (on Chromosome 14) is detailed in Table 2. Droplets (10,000-20,000 per sample) were generated using the QX200 Droplet Generator (Bio-Rad), and PCR amplification was performed in a thermal
<figure><img src="image_4.png" /><figcaption>Fig. 1.</figcaption></figure>
cycler. Following amplification, droplets were read using the QX600 Droplet Reader (Bio-Rad), and data were analyzed with QuantaSoft Analysis Pro software (Bio-Rad). PSEN1 levels were quantified as the ratio of PSEN1- to SF3B1-positive droplets.

### 3.4. Karyotype and genetic recurrent abnormalities analysis

Karyotype and genomic stability were assessed by detection of recurrent genetic abnormalities using, respectively, iCS-Karyo and iCS-digital™ PSC tests, provided as a service by Stem Genomics (https://www/stemgenomics.com/), as previously described (Assou et al., 2020; Clua Provost et al., 2023).

### 3.5. Trilineage differentiation

The trilineage differentiation kit (Stem Cell Technologies) was performed following the manufacturer's instructions.

<table><caption>**Table 1**

Characterization and validation.</caption><thead><tr><th>Classification<br>(optional italicized)</th><th>Test</th><th>Result</th><th>Data</th></tr></thead><tbody><tr><td>Schematic of a transgene/genetic modification</td><td>Schematic illustrating the structure and location of the introduced genetic modification</td><td>Knock-In to correct the heterozygote PSEN1 mutation</td><td>Fig. 1 panel A</td></tr><tr><td>Morphology</td><td>Photography</td><td>Typical iPS cell-like morphology</td><td>Figure panel C</td></tr><tr><td>Pluripotency status evidence for the described cell line</td><td>Qualitative analysis by immunostaining and imaging</td><td>Cells are positive for pluripotency markers: OCT3/4, SOX2, NANOG</td><td>Figure panel E</td></tr><tr><td>Karyotype</td><td>Karyotype (G-banding)<br>iCS-digital™ PSC test</td><td>N/A<br>46,XY<br>Resolution 300-500<br>No CNVs detected for the 24 probes tested</td><td>N/A<br>Figure panel D<br>Supplementary Data S4</td></tr><tr><td>Genotyping for the desired genomic alteration/allelic status of the gene of interest</td><td>PCR across the edited site or targeted allele-specific PCR</td><td>PCR + Sanger sequencing<br>Confirmation of the correction.</td><td>Figure panel B<br>Sanger sequencing chromatograms (primary data *.ab1 in supplementary files).<br>Digested PCR products on Supplementary S1.<br>Supplementary Data S6</td></tr><tr><td>Verification of the absence of random plasmid integration events</td><td>Evaluation of the (homo-/hetero-/hemi-) zygous status of introduced genomic alteration(s)<br>Transgene-specific PCR (when applicable)</td><td>ddPCR + Sanger sequencing<br>Absence of allelic dropout.<br>Homozygous status for codon 650 (wild-type / corrected).<br>N/A</td><td>N/A</td></tr><tr><td>Parental and modified cell line genetic identity evidence</td><td>PCR</td><td>PCR specific for puromycin resistance gene of SpCas9(BB)-2A-Puro plasmid.<br>No detection.</td><td>Supplementary Data S3</td></tr><tr><td>Mutagenesis / genetic modification outcome analysis</td><td>Sequencing (genomic DNA PCR or RT-PCR product)<br>PCR-based analyses<br>Southern Blot or WGS; western blotting (for knock-outs, KOs)</td><td>STR Profiling for amelogenin and 15 loci (D8S1179, D21S11, D3S1358, D5S818, D13S317, D7S820, D16S539, D2S1338, D19SS433, vWA, Th01, TPOX, D18S51, CSF1PO? FGA).<br>STR profile analysis reveals a 100 % match between edited and parental cell lines.</td><td>Supplementary file 1</td></tr><tr><td>Off-target nuclease activity analysis</td><td>PCR across top 6 predicted top likely off-target sites</td><td>Confirmation of correction of the heterozygous mutation G217D in the PSEN1 gene.<br>Absence of unintended allelic dropout (ddPCR)<br>N/A</td><td>Figure panel B<br>Supplementary Data S6<br>N/A</td></tr><tr><td>Specific pathogen-free status</td><td>Mycoplasma</td><td>No off-target effect occurred in the top predicted sites.</td><td>Supplementary Data S5</td></tr><tr><td>Multilineage differentiation potential</td><td>Directed differentiation</td><td>Negative<br>Cells demonstrated their ability to differentiate into derivatives of all 3 germ layers. Expression of ectodermal (Nestin, Pax6), endodermal (Sox17, FOXA2) and mesodermal (Brachyury, A-SMA) markers, by immunofluorescence.</td><td>Supplementary Data S2<br>Figure panel E</td></tr><tr><td>Outcomes of gene editing experiment<br>(OPTIONAL)</td><td>Brief description of the outcomes in terms of clones generated/ establishment approach/screening outcomes</td><td>Numbers of clones: total established, unedited, targeted by HDR, editing by-products (NHEJ/indels), etc.</td><td>37 transfected clones, 5 clones screened based on digestion profile, 2 corrected mutation confirmed</td></tr><tr><td>Donor screening (OPTIONAL)</td><td>HIV 1 + 2 Hepatitis B, Hepatitis C</td><td>N/A</td><td>N/A</td></tr><tr><td>Genotype – additional histocompatibility info<br>(OPTIONAL)</td><td>Blood group genotyping<br>HLA tissue typing</td><td>N/A<br>N/A</td><td>N/A<br>N/A</td></tr></tbody></table>

### 3.6. Immunofluorescence

Cells were cultured on geltrex-coated coverslips and fixed using 4 % paraformaldehyde for 15 min before immunostaining. For intracellular labelling, cells were first permeabilized with DPBS- 0.5 % Triton X-100 for 3 x 10 min. Non-specific binding sites were blocked with 1 h incubation in DPBS-0.2 % BSA. Antibodies were diluted in blocking buffer (Table 1 for dilutions). Primary antibodies were incubated overnight in humidified chamber, followed by 3 washes in blocking buffer. Secondary antibodies were then incubated during 1 h. Nuclei were stained with DAPI (Thermo Fischer Scientific), diluted at 1:1000 in DPBS, during 30 min. Coverslips were washed in DPBS and water, before mounting with FluorSave (Millipore). Cells were imaged with the epifluorescence microscope Upright Zeiss Apotome.

### 3.7. Cell Line Authentication

Genomic identity was assessed by Short Tandem Repeat (STR)
<table><caption>**Table 2**
Reagents details.</caption><thead><tr><th colspan="3">Antibodies and stains used for immunocytochemistry/flow-cytometry</th></tr><tr><th></th><th>Antibody</th><th>Dilution</th><th>Company Cat # and RRID</th></tr></thead><tbody><tr><td rowspan="3">Pluripotency Markers</td><td>Mouse anti-OCT3-4 (C-10)</td><td>1:500</td><td>Santa Cruz</td></tr><tr><td>Rabbit anti-SOX2</td><td>1:100</td><td>Biotechnology Cat# sc-5279, RRID:AB_628051</td></tr><tr><td>Goat anti-NANOG</td><td>1:20</td><td>Bethyl Cat# 301-739A, RRID:AB_1211354</td></tr><tr><td rowspan="6">Differentiation Markers</td><td>Rabbit anti-Nestin</td><td>1:200</td><td>R and D Systems Cat# AF1997, RRID: AB_355097</td></tr><tr><td>Mouse anti-PAX6</td><td>1:300</td><td>Proteintech Cat# 19483-1-AP, RRID: AB_1064440</td></tr><tr><td>Rabbit anti-HNF3beta/FOXA2</td><td>1:100</td><td>Millipore Cat# MAB5552, RRID: AB_2159810</td></tr><tr><td>Mouse anti-SOX17</td><td>1:50</td><td>Millipore Cat# 07-633, RRID:AB_390153</td></tr><tr><td>Rabbit anti-alpha smooth muscle Actin</td><td>1:200</td><td>R and D Systems Cat# MAB1924, RRID: AB_2195646</td></tr><tr><td>Goat anti-Brachyury</td><td>1:10</td><td>Abcam Cat# ab5694, RRID:AB_2223021</td></tr><tr><td rowspan="4">Secondary antibodies</td><td>Donkey Anti-Mouse IgG, Alexa Fluor 555-conjugated</td><td>1:1000</td><td>Molecular Probes Cat# A21424, RRID: AB_141780</td></tr><tr><td>Goat Anti-Rabbit IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular Probes Cat# A-11008, RRID: AB_143165</td></tr><tr><td>Donkey Anti-Rabbit IgG, Alexa Fluor 488-conjugated</td><td>1:1000</td><td>Molecular Probes Cat# A21206, RRID: AB_2535792</td></tr><tr><td>Donkey Anti-Goat IgG, Alexa Fluor 555-conjugated</td><td>1:1000</td><td>Molecular Probes Cat# A21432, RRID: AB_2535853</td></tr><tr><td>Nuclear stain</td><td>DAPI</td><td>5 µg/mL</td><td>Thermo Fischer Scientific cat # D3571</td></tr><tr><td colspan="4"><strong>Site-specific nuclease</strong></td></tr><tr><td>Nuclease information</td><td>Nuclease type/ version</td><td></td><td>SpCas9</td></tr><tr><td>Delivery method</td><td>Electroporation (parameters)</td><td></td><td>Neon™ Transfection System (Invitrogen) 1,100 V 30 ms 1 pulse</td></tr><tr><td>Selection/ enrichment strategy</td><td>Selection cassette</td><td></td><td>Puromycin resistance</td></tr><tr><td colspan="4"><strong>Primers and Oligonucleotides used in this study</strong></td></tr><tr><td>Episomal Plasmids (qPCR or RT-PCR)</td><td>N/A</td><td></td><td>N/A</td></tr><tr><td>Pluripotency Markers (qPCR)</td><td>N/A</td><td></td><td>N/A</td></tr><tr><td>SgRNA design</td><td>Primers</td><td></td><td>Forward: cac cGA GAT CCA CTT CGA CTC CAG C<br>Reverse: aaa cGC TGG AGT CGA AGT GGA TCT C</td></tr><tr><td>House-Keeping Genes (qPCR)</td><td>GAPDH</td><td></td><td>Forward: CGC TCT CTG CTC CTC CTG TT<br>Reverse: CCA TGG TGT CTG AGC GAT GT</td></tr><tr><td>Genotyping (desired allele/ transgene presence detection)</td><td>PCR specific for the targeted allele</td><td></td><td>Primers to amplify 300 bp amplicon having mutation in the middle of the product<br>Forward: TGG TGA AAA TTA TTG TAC ATC TTT TAA AAT CT<br>Reverse: TGA CAA ACA AAT TAT CAG TCT TGG G</td></tr></tbody></table>

<table><caption>**Table 2 (continued)**</caption><thead><tr><th colspan="4">Antibodies and stains used for immunocytochemistry/flow-cytometry</th></tr><tr><th></th><th>Antibody</th><th>Dilution</th><th>Company Cat # and RRID</th></tr></thead><tbody><tr><td>Targeted mutation analysis/ sequencing</td><td>Sequencing data from both alleles</td><td></td><td>Sanger sequencing chromatograms in Supplementary files.</td></tr><tr><td>Potential random integration-detecting PCRs</td><td>Puromycin resistance cassette</td><td></td><td>Primers described above.<br><strong>Forward:</strong> ATG ACC GAG TAC AAG CCC AC<br><strong>Reverse:</strong> GAG GCC TTC CAT CTG TTG CT</td></tr><tr><td>gRNA oligonucleotide/crRNA sequence</td><td>crRNA sequence</td><td></td><td>A GAT CCA CTT CGA CTC CAG C [AGG]</td></tr><tr><td>Genomic target sequence</td><td>Including PAM and other sequences likely to affect UCN activity</td><td></td><td>PSEN1 c.650G > A p.Gly217Asp, exon 7, chromosome 14<br>TG ATT TCC ATT CAC TGG AAA GAT CCA CTT CGA CTC CAG CAG GCA TAT CTC ATT ATG ATT AGT GCC CTC ATG GCC CTG GTG TTT ATC AAG TAC CTC CCT GAA TGG ACT GCG TGG CTC ATC TTG GCT GT<br><a href="https://crispor.gi.ucsc.edu/crispor.py?batchId = c3liuHesvcwe0zUawhhg">https://crispor.gi.ucsc.edu/crispor.py?batchId = c3liuHesvcwe0zUawhhg</a></td></tr><tr><td>Bioinformatic gRNA on- and off-target binding prediction tool used, specific sequence/outputs link(s)</td><td>CRISPOR, CHOP CHOP and for gRNA design</td><td></td><td>CRISPOR for off-target sites prediction</td></tr><tr><td>Primers for top off-target mutagenesis predicted site sequencing</td><td>OT1 – F&amp;R MTSU2<br>OT2 – F&amp;R RP11-540 N6.1<br>OT3 – F&amp;R PRDM16<br>OT4 – F&amp;R M*APK6P-RN7SKP32<br>OT5 – F&amp;R ACA64-CTD-2194 M22.1<br>OT6 – F&amp;R EPT1</td><td></td><td><strong>Forward:</strong> GCC AGT TCT GAA GGC ATC TC<br><strong>Reverse:</strong> CCT CTC CAA GGC ACA AAG TC<br><strong>Forward:</strong> CAAACGTCTGGCTTCCATTT<br><strong>Reverse:</strong> TTGCATTTCTGTGAGCCAAG<br><strong>Forward:</strong> AGGGCAGATTTGATGAAACG<br><strong>Reverse:</strong> CAAGCTTCTTGCTCACCAGA<br><strong>Forward:</strong> ATTATTTGGGGGCTGCTTTT<br><strong>Reverse:</strong> GTGCTGAACTAGGCCCAGAG<br><strong>Forward:</strong> TTTGGAGCTGTGAGTTGTGC<br><strong>Reverse:</strong> GACTGCGATCCTTAGGCAAG<br><strong>Forward:</strong> CTTAAGCGAGGGGGTTGTGT<br><strong>Reverse:</strong> CCATGTGAATGGTCTGTGGA<br>AC AGC CAA GAT GAG CCA CGC AGT CCA TTC AGG GAG GTA CTT GAT AAA CAC GAG GGC CAT GAG GGC ACT AAT CAT AAT GAG ATA TGC TTG CTG GAG TCG AAG TGG ACC TTT CCA GTG AAT GGA AAT CA</td></tr><tr><td>ODNs/plasmids/ RNA molecules used as templates for HDR-mediated site-directed mutagenesis.</td><td>ssODN sequence<br>The corrected single nucleotide is in red, the mutated PAM sequence is underlined.</td><td></td><td></td></tr><tr><td>ddPCR for allele copy number assay</td><td>Probe – PSEN1 Primers – F&amp;R – PSEN1<br>Probe &amp; primers – SF3B1</td><td></td><td>ATGGCCCTGGTGTTTATCAAGTAC – HEX<br><strong>Forward:</strong> TTTTGGTGTGGTGGGAATG<br><strong>Reverse:</strong> CTGAAATCACAGCCAAGATG<br>BioRad #10042958 – FAM</td></tr></tbody></table>

analysis, provided as a service by Stem Genomics (https://www.stemgenomics.com/), on genomic DNA extracted from parental and modified cell lines.

### 3.8. Mycoplasma

The absence of mycoplasma contamination was performed with MycoAlert® Mycoplasma detection assay (Lonza).
CRediT authorship contribution statement

**Coralie Clua Provost:** Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Louise Greetham:** Investigation. **Cécile Monzo:** Investigation. **Arnaud Monteil:** Methodology. **Anne Rovelet-Lecrux:** Resources. **Sylvain Lehmann:** Validation, Formal analysis. **David Wallon:** Resources. **Valentin Garcia:** Methodology, Formal analysis. **Hélène Hirbec:** Supervision, Formal analysis. **Emmanuel Nivet:** Writing – review & editing, Validation, Supervision, Methodology, Investigation, Formal analysis, Conceptualization. **Carole Crozet:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Methodology, Funding acquisition, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.scr.2025.103780.

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