Generation of heterozygous SAMD9 CRISPR/Cas9-edited iPSC line (ESi086-A-3), carrying p.I1567M mutation

Germline SAMD9 mutations are one of the most common alterations that predispose to pediatric myelodysplastic syndrome (MDS), a clonal disorder characterized by ineffective hematopoiesis, increasing the risk of developing acute myeloid leukemia (AML). Up to date, a disease model to study the role of SAMD9 mutation in MDS is still lacking. Here, we have generated a human induced pluripotent stem cell (hiPSC) line carrying SAMD9 mut (p.I1567M


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Induced Pluripotent Stem Cells (iPSCs) carrying heterozygous SAMD9 mutation (p.I1567M), offer an unprecedent opportunity to generate unlimited iPSC-derived blood progenitors in vitro, bypassing the necessity of primary patient samples. Moreover, this human-based SAMD9 model will allow a deep understanding of myelodysplastic predisposition susceptibility related to SAMD9-germline mutation. Table 1.

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Sterile alpha motif domain protein 9 (SAMD9) is a highly conserve genes where missense mutations are associated with predisposition to familial MDS and increased the risk to develop acute myeloid leukemia (AML) (Schwartz et al., 2021). SAMD9 have a common expression in the human tissue where it regulates cell proliferation and apoptosis (Li et al., 2007). SAMD9 function is currently cryptic but it has been shown an antiproliferative potential as well as a tumour suppressor function, among others (Sahoo et al., 2021). Figure 1 Individuals carrying a germline heterozygous SAMD9 mutation often suffer from a transient aplasia, cytopenia, MIRAGE syndrome and monosomy 7 among others (Davidsson et al., 2018). Nowadays, the hematopoietic stem cell transplantation (HSCT) is the only curative treatment, and the 5-year overall survival is ~84% in cases with normal karyotype. However, in patients with abnormal karyotype as monosomy 7 the overall survival is 70-80% (Sahoo et al. Nat Med 2021). The understanding of the role of germline SAMD9 mutations in familial MDS/ AML will be critical to elucidate the molecular mechanisms of disease progression. Therefore, a reliable and trustworthy disease model is required. The identified SAMD9 mutations are described as missense, inframe deletion, frameshift, or nonsense. The germline mutations in SAMD9 resulted in gain of function (GOF), promoting cell apoptosis and a cell growth-suppressive effect compared to the non-mutated cells (Sahoo et al., 2021). Consequently, taking advantage of precise gene editing tools, we have generated human iPSCs carrying the p.I1567M SAMD9 mutations described in familial MDS patients (Sahoo et al., 2021).
Using a pre-designed sgRNA, targeting p.I1567M mutation, the CRISPR/Cas9 system was applied on healthy iPSC line (CBI08-3F-4). The iPSCs were nucleofected with Cas9 protein, the sgRNA together with a ssODN donor template carrying the mutation c.4701A>G. Right after, we performed a positive clone screening selection by Restriction Length Polymorphism (RFLP) by digestion with NlaIII and confirmed them by Sanger sequencing.
After the selection and expansion of the clone carrying the p.I1567M mutation, a karyotype analysis was performed to avoid chromosomic abnormalities. Concluding that the engineered hiPSC line ESi086-A-3 has a normal 46XY karyotype, showing alkaline phosphatase (AP) activity which is a marker to identify pluripotent stem cells. Immunofluorescence and flow cytometry analysis revealed expression of pluripotency markers such as, NANOG, OCT4, SSEA4, TRA-1-81 and TRA-1-60. Additionally, the gene expression of OCT4, SOX2, NANOG, CRIPTO and REX1 was confirmed by RT-PCR. The SAMD9 p.I1567M line has high capacity to generated embryoid bodies (EBs), which could be differentiated into derivatives of the three embryonic germ layers: being positive for TUJ1 and GFAP in ectoderm, α-fetoprotein (AFP) and FoxA2 in endoderm, and CD45/CD34 in mesoderm The iPSC line identity was confirmed by short tandem repeat analysis (STR) and compared with the original parental CBiPS8-3F-4 clone. The cell line was free from mycoplasma contamination as determined by PCR.

Human iPSC cultures
hiPSC line is maintained in a feeder-free culture system on Matrigel  ATACCCATCACTCCCGCTTTTTTAGGTCAACTTAGAAGTGGCAGAAGCATgGAGAAaGTGTCTTTTTACCTGGGATTTTCCATTGGAGGCCCACTTGCTT (BD Biosciences, MA)-coated 60-mm plates with mTeSR1 medium (StemCell Technologies) at 37 • C, 5% CO 2 & 20% O 2 . Culture medium was changed daily, and the cell was passaged weekly by EDTA dissociation (PBS + 0.5mM EDTA).

CRISPR/Cas9 gene editing
The CRISPR tool (https://bioinfogp.cnb.csic.es/tools/breakingcas/) was used in the sgRNA design. A high probability to target the region of interest and low probability to generate off-targets gRNA sequence was selected. Rock inhibitor (Y-27632, 10µM) was added to the iPSCs 3 hours before nucleofection. 100 pmol Alt-R® CRISPR-Cas9 (IDT) was incubated with 120 pmol Alt-R® CRISPR-Cas9 sgRNA (IDT) at 25 • C for 10 min. 4uM of ssODN was added to the RNP complex prior the nucleofection. 200.000 cells were dissociated with Accutase (Gibco), washed twice with PBS without Ca and Mg and resuspended with 20ul of P3/ S1Buffer. RNP complex + ssODN was added to the cell pellet and transferred to the 20ul cuvette. Cells were nucleofected with 4-D Nucleofector System (Lonza) using the CA-137 program. Nucleofected cells were cultured in a 12 well plate, with mTSR1 and 10uM of Y-27632. After 72h of recovery, 1000 cells were seeded at a single cell level in a 100 mm plate to form single-cell colony. Genotyping was performed by PCR, RFLP and Sanger sequencing in single cell colonies to analyse the gene mutation.

Alkaline Phosphatase
iPSCs were fixed with 4% paraformaldehyde (PFA) for 1 min, washed with PBS and incubated with AP solution (Sigma) for 20 minutes.

Flow Cytometry
iPSCs at passage 38 were dissociated as single-cell using Accutase (Gibco). Cells were stained with FACS antibodies for 15 min at RT in the dark. Gallios Flow Cytometer (Beckman Coulter) with the appropriate laser and filters sets was used to ran samples. The positive population was gated using Kaluza Analysis Software (Beckman Coulter).

Quantitative RT-PCR
mRNA was isolated from iPSCs at passage 39 using the standard Maxwell® RSC Instrument for automated RNA extraction and following manufacturer's protocol. SuperScript III (Invitrogen) was used to generate cDNA. For qRT-PCR, SYBR green (Life technologies) was used. Primer sequences are listed in Table 2. Ct values were normalized using GAPDH as housekeeping gene. Assays were run on the ABI PRISM 7900HT system (Applied Biosystems) and data were analysed with the 2-ΔCt method.

Sequencing
Genomic DNA was isolated using QIAamp DNA Mini Kit (QIAGEN) according to manufacturer's protocol. PCR was performed with the primers GATA2-gDNA-PCR ( Table 2) with GoTaq Flexi DNA Polymerase (Promega) with the following protocol: 95 • C for 5 min, 35 cycles of 95 • C for 30s, 61 • C for 30s, 72 • C for 60s and 72 • C for 7min. PCR products were purified with DNA Clean & Concentrator-5 (Zymo Research) and sent for sequencing to Eurofins. Genetic alterations were identified using Benchling.

Mycoplasma
Supernatant of confluent iPSCs at passage 37 was harvested. Venor GeM Classic Mycoplasma Detection Kit (Minerva Biolabs) was used following manufacturer's protocol.

Author contributions
J.P. designed the study, carried out the experiments, analyzed the data and wrote the manuscript; J.C. designed the study; J.C. carried out the experiments; A.G. and D.R.M analyzed and discussed the data and wrote the manuscript; all authors approved the final version of the manuscript

Abnormal karyotype
The cell line presents a normal karyotype

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.