CRISPR-based screening identifies XPO7 as a positive regulator of senescence

WT hESCs (line H9, WiCell Research) and XPO7 hESCs were maintained on mitomycin C-inactivated mouse embryonic fibroblasts (MEFs) with ESC culture medium (80% DMEM/F12 (Gibco), 20% Knockout Serum Replacement (Gibco), 1% non-essential amino acids (NEAA, Gibco), 1% GlutaMAX (Gibco), 1% penicillin/streptomycin (Gibco), 55 μmol/L β-mercaptoethanol (Invitrogen), and 10 ng/mL fibroblast growth factor 2 (FGF2, Joint Protein Central)); hESCs were also cultured on plates coated with Matrigel (BD Biosciences) in the mTeSR medium (STEMCELL Technologies, Vancouver).

the sgRNAs that rejuvenate hMSCs to become dominant. With cell culturing for 36 days (RS hMSC) or 46 days (WS hMSC) or 54 days (HGPS hMSC), cells were harvested and the genomic DNA was extracted by DNeasy Blood & Tissue Kit (QIAGEN) following the manufacturer's protocol. Then the genomic DNA was sequenced, and the initial point was assessed by plasmid sequencing.
PCR was performed by PrimeSTAR HS DNA Polymerase (Takara) following the manufacturer's instructions. A two-step PCR process was used to amplify inserted sgRNA sequences, appended molecular barcodes and Illumina adapter sequences. The reaction conditions of the first PCR step: a) denaturation at 98°C, 2 min; b) 19 cycles at 98°C, 10 s; c) annealing at 58.0°C, 30 s; d) extension at 72°C, 40 s; e) final single extension step at 72°C, 5 min. After that, the amplicons were purified by C1 beads (Thermo Fisher Scientific) and then used as the template for the second PCR step. Illumina HiSeq 2500 adapter and barcode sequences were appended to the primers used in the second PCR step. The reaction conditions were the same as the first PCR step except for 14 cycles in step b). The primers are listed in Table S3.

Ultraviolet (UV)-, Oncogene-, and H 2 O 2 -induced cellular senescence
Cellular senescence induced by different cellular stresses was performed as previously described . For UV-induced cellular senescence, WT hMSCs were irradiated at 10 J/m 2 by XL-1000 UV Crosslinker (Spectronics Corporation). For oncogene-induced cellular senescence, WT hMSCs were transduced with pBABE-puro-Ras V12 retrovirus. For H 2 O 2 -induced cellular senescence, we treated WT hMSCs with 30 μM H 2 O 2 for 24 h. When the cells reached 95% confluence, cells were passaged and processed for phenotypic analyses.

SA-β-gal staining
SA-β-gal staining was performed as previously described (Kubben et al., 2016). Cells were fixed with the fixation buffer (PBS with 2% formaldehyde and 0.2% glutaraldehyde) for 5 min at room temperature (RT). Then, cells were stained by fresh staining buffer containing X-gal at 37°C overnight. The percentage of positive cells was calculated and analyzed by ImageJ. Three biological replicates are performed for each group (n = 3).

Clonal expansion assay
Clonal expansion assay in hMSCs was performed as previously described (Li et al., 2020). 2,000 cells were seeded in a 12-well plate and cultured for about 2 weeks. Then, cells were fixed by 4% paraformaldehyde (PFA) for 30 min, followed by staining with 0.2% crystal violet for 1 hr at RT. Relative cell density was quantified with ImageJ. Three biological replicates are performed for each group (n = 3).

Western blot analysis
Cells were lysed in SDS (Sigma-Aldrich) buffer as previously described (Bi et al., 2020), then a BCA kit (Thermo Fisher Scientific) was used to measure the protein concentration. 20 μg protein was subjected to SDS-PAGE and electrotransferred to PVDF membranes (Millipore). Then the membranes were blocked by 5% non-fat milk. After that, the membranes were incubated with specific primary antibodies overnight at 4°C , followed by HRP-conjugated secondary antibodies. The quantification of each image was detected by the ChemiDoc XRS+ system (Bio-Rad). GAPDH was used as a loading control. Three independent experiments were performed (n = 3) for each assay.

Immunofluorescence staining
Immunofluorescence staining was performed as previously described (Liu et al., 2012). Cells seeded on coverslips were fixed by 4% PFA for 30 min at RT, followed by washing with PBS and permeabilizing with 0.4% Triton X-100 (Sigma-Aldrich) for 10 min. Then 10% donkey serum was used to block cells for 1 hr at RT, and the indicated primary antibodies were incubated overnight at 4°C . After that, cells were washed by PBS and incubated with secondary antibodies and Hoechst33342 (Thermo Fisher Scientific) for 1 hr at RT. Images were taken by Zeiss LSM900 confocal microscope. For quantification of Ki67-positive cells, three biological replicates were set for each group (n = 3), and for quantification of H3K9me3 fluorescence intensity in each group was performed from 300 cells (hMSC) or 150 cells (primary fibroblast) from 3 biological replicates.

Cell cycle analysis
Cells were fixed with 70% ethanol overnight at -20°C. After that, cells were washed with PBS, permeabilized with 0.1% Triton X-100 and incubated with 0.2 mg/mL RNase A and stained with 0.02 mg/mL propidium iodide at 37°C for 30 min. The cell cycle analysis was conducted by BD LSRFortessa Cell Analyzer. Three biological replicates are performed for each group (n = 3).

Enzyme-Linked Immunosorbent Assay (ELISA)
The IL-6 secretion of hMSCs was detected by an ELISA kit (Biolegend,430504) following the manufacturer's instructions. In brief, the medium was collected and incubated in an anti-IL6 antibody-coated plate. After that, Avidin-HRP, freshly mixed TMB substrate, and stop solution were added. Then the plate was measured at 450 nm. IL-6 levels were normalized to the corresponding cell numbers. Three biological replicates were performed for each group (n = 3)

RNA and DNA analyses
Total RNA was extracted by TRIzol™ (Thermo Fisher Scientific), after that GoScript™ Reverse Transcription System (Promega) was used to generate cDNA. After that, RT-qPCR was performed using the qPCR Mix (TOYOBO) in a CFX384 Real-Time system (Bio-Rad). GAPDH was used as an internal control. Four biological replicates were performed for each group (n = 4). DNeasy Blood & Tissue Kit (QIAGEN) was used to extract the genomic DNA, and the PCR amplification was performed by PrimeSTAR DNA Polymerase. The primers used for qPCR and PCR are listed in Table S3.
For RNA-seq, 1~2 µg of total RNA was quantified using the fragment analyzer (Advanced Analytical) and sequenced with Novaseq 6000.

Co-immunoprecipitation (Co-IP)
Co-IP assay was performed as previously reported (Diao et al., 2021). HEK293T cells were transfected with plasmids expressing Flag-XPO7 or Flag-Luc. Then cells were lysed in CHAPS lysis buffer (120 mM NaCl, 0.3% CHAPS, 40 mM HEPES (pH 7.5), 1 mM EDTA, and protease inhibitor cocktail (Roche)) at 4°C for 2 hr and centrifuged at 12,000 g, 4°C for 30 min. The supernatants were incubated with anti-FLAG Affinity Gel (Sigma, A2220) at 4°C overnight. FLAG peptides were used to obtain the XPO7-interacting protein complexes, and processed for western blot or LC-MS/MS analysis. For endogenous co-IP assays, after cell lysis, the supernatants were mixed with indicated antibodies and rotated at 4°C overnight. After that, the supernatants were incubated with beads at 4°C for 3 hr. Then, the immunocomplexes were washed three times with CHAPS lysis buffer and processed for western blot.

LC-MS/MS analysis
The eluted proteins obtained by Co-IP were subjected to 10% SDS-PAGE gel and stained by Coomassie brilliant blue. Then the gel bands were excised, cut into small plugs, dehydrated (100% acetonitrile), reduced (10 mM DTT in 25 mM NH 4 HCO 3 at 56°C for 45 min) and alkylated (40 mM iodoacetamide in 25 mM NH 4 HCO 3 at RT for 45 min in the dark). After that, the gel plugs were dried and digested with sequencing-grade modified trypsin (40 ng per band) in 25 mM NH 4 HCO 3 at 37°C overnight. Formic acid was used to terminate the enzymatic reaction. The nanoLC-MS/MS experiments were performed on Q Exactive mass spectrometer (Thermo Scientific). Then, the raw data from Q Exactive analysis were analyzed with Proteome Discovery (2.2.0.388) using the Sequest HT search engine for protein identification. The data were searched against the UniProt human protein database (update-180920). False discovery rate (FDR) < 1% was set as the threshold for protein identification, and the peptide confidence was set to high for peptide filtering. XPO7-interacting proteins are listed in Table S2.

CRISPR-based screening analysis
Sequencing data were analyzed with MAGeCK (version 0.5.9.2). Read counts were initially obtained from samples by "count" subcommand in MAGeCK, in which the data quality was accessed by fastqc, and reads with high-quality were further mapped to the screening library. Then, compared with the count of baseline library, the positively and negatively selected sgRNAs were identified by the "test" subcommand. Finally, sgRNAs with P-value < 0.05 were considered as significant and ranked by RRA (robust rank aggregation) score in positive selection.

RNA-seq analysis
For RNA-seq raw data, sequences with adaptors and low-quality reads were removed by Trim Galore (version 0.4.5). Clean data was mapped to the human hg19 reference genome by STAR (version 2.7.1a) software with default parameters (Dobin et al., 2013). The reads mapped to each gene were calculated using featureCounts (version 2.0.1) (Liao et al., 2014). Differentially expressed genes (DEGs) were calculated using the R package DESeq2 (version 1.30.1) with the cutoff of adjusted P-value less than 0.05 and |log 2 (fold change)| more than 0.5 (Love et al., 2014). Gene Ontology (GO) terms and pathways enrichment analysis was performed by Metascape (Zhou et al., 2019).

Copy number variation (CNV) analysis
For CNV data analysis, raw reads were trimmed with TrimGalore (version 0.4.5), then aligned to the human hg19 reference genome by Bowtie2 (version 2.4.2) (Langmead and Salzberg, 2012). The mapping reads are counted for each 500-kb bin using readCounter function of hmmcopy_utils (https://github.com/shahcompbio/hmmcopy_utils). The R/Bioconductor package HMMcopy (version 1.26.0) was used to correct copy number, GC content and mappability.

Data Availability
The screening and transcriptomic data obtained in the study have been deposited in the Genome Sequence Archive in the National Genomics Data Center, Beijing Institute of Genomics (China National Center for Bioinformation) of the Chinese Academy of Sciences (Chen et al., 2021), under accession number HRA003304 and HRA003369. The LC-MS/MS data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository (Ma et al., 2019) under accession number PXD037890. (E) ELISA analysis for the secretion of interleukin-6 (IL-6) in RS hMSCs after CRISPR-mediated knockout (CRISPRko) of XPO7. Data are presented as the mean ± SEM. n = 3 biological replicates. *, P < 0.05. (F) Immunofluorescence analysis of H3K9me3 in RS hMSCs after CRISPR-mediated knockout (CRISPRko) of XPO7. Scale bars, 20 μm. The white arrowheads denote the cells with increased H3K9me3 signals. Data are presented as the mean ± SEM. n = 300 cells from three biological replicates. ***, P < 0.001.

Supplementary Table Legends
Table S1. Gene rank of CRISPR/Cas9-based LOF screening and differentially expressed genes (DEGs) in XPO7 −/− hMSCs. Table S2. Candidate XPO7-interacting proteins identified by LC-MS/MS. Table S3. Sequence information of sgRNAs and primers used for CRISPR/Cas9-based screen library construction, gene editing, plasmid construction, RT-qPCR analysis as well as the donor information of human primary fibroblast.