Also in the Article

Female 1.8 XX mESCs carry a homozygous insertion of 7xMS2 repeats in Xist exon 7 and are a gift from the Gribnau lab [11]. Several clones with XX or XO genotype (loss of one X chromosome) were generated through sub-cloning of the parental XX cell line. Female TX1072 ESCs carry a doxycycline responsive promoter in front of the Xist gene on one X chromosome and have been described previously [11]. For detailed information on the cell lines, refer to Additional file 9: Table S8. Low-passage Hek293T cells were a kind gift from the Yaspo lab.

The 1.8 SRE-Elk cell line was generated by lentiviral transduction of 1.8 XX mESCs with the pLenti-SRE/Elk-GFP-PEST-Hygro plasmid (Additional file 9: Table S8) followed by Hygromycin (250 ng/μl, VWR) selection. Single clones were picked and expanded and GFP expression confirmed via flow cytometry.

To identify Klhl13 interaction partners, female K13-HOM mESCs (Clone 2) were transduced with the lentiviral plasmids pLenti-PGK-Degron-GFP-Blast, pLenti-PGK-Degron-GFP-Klhl13-Blast, pLenti-PGK- GFP-Blast, and pLenti-PGK-GFP-Kelch plasmids (Additional file 9: Table S8) and selected using blasticidin (5 ng/μl, Roth). Protein expression was assessed via immunoblotting.

In 1.8-Nanog-mCherry and 1.8-Esrrb-mCherry reporter lines, the C-Terminus of the coding sequences of the Nanog or Esrrb genes, respectively, is tagged with the fluorescent protein mCherry, separated by a P2A self-cleaving peptide.

Cell lines over-expressing Klhl13 and Dusp9 via the CRISPRa Suntag system were generated by lentiviral transduction of E14-STN cells, which express the CRISPR activating Sun-Tag system [55] under a doxycycline-inducible promoter (kind gift from Navarro lab, [56]), with plasmids carrying sgRNAs targeted to the respective promoters or non-targeting controls (pU6-Klhl13.1-EF1Alpha-puro-T2A-BFP, pU6-Klhl13.2-EF1Alpha-puro-T2A-BFP, pU6-Dusp9.1-EF1Alpha-puro-T2A-BFP, pU6-Dusp9.2-EF1Alpha-puro-T2A-BFP, pU6-NTC.1-EF1Alpha-puro-T2A-BFP, pU6-NTC.2-EF1Alpha-puro-T2A-BFP, Additional file 9: Table S8; Additional file 1: Figure S3A) followed by puromycin selection (1 ng/μl, Sigma).

Dusp9 and Klhl13 heterozygous (HET) and homozygous (HOM) together with Dusp9 and Klhl13 double heterozygous mutant cell lines were generated via Cas9-mediated genome editing (see below) of 1.8 XX mESCs.

Cell lines for Klhl13 and Dusp9 knock-down were generated by lentiviral transduction of the 1.8 XX SP107 cell line (Clone A2, see below) with plasmids carrying sgRNAs targeting their respective promoters or a non-targeting control (SP199_multi_Dusp9_CRISPRi, SP199_multi_Klhl13_CRISPRi and SP199_multi_NTC1, Additional file 9: Table S8). Similarly, cell lines for Alg13, Cct3, Larp1, Peg10, and Scml2 knock-down and over-expression were generated by lentiviral transduction of the 1.8 XX K13-HOM SP107 and 1.8 XX SP106 cell line (see below), respectively, with plasmids carrying sgRNAs targeting their respective promoters (SP199_multi_Alg13_CRISPRi, SP199_multi_Cct3_CRISPRi, SP199_multi_Larp1_CRISPRi, SP199_multi_Peg10_CRISPRi, SP199_multi_Alg13_CRISPRa, SP199_multi_Cct3_CRISPRa, SP199_multi_Larp1_CRISPRa, SP199_multi_Peg10_CRISPRa, SP199_multi_Peg10_CRISPRa_2 and SP199_multi_Scml2_CRISPRa, SP199_multi_NTC1 and SP199_multi_NTC2, Additional file 9: Table S8). All cell lines were selected with puromycin (1 ng/μl, Sigma) for stable sgRNA integration.

All mESC lines were grown without feeder cells on gelatin-coated flasks (Millipore, 0.1%) in serum-containing ES cell medium (DMEM (Sigma), 15% FBS (PanBiotech), 0.1 mM β-Mercaptoethanol (Sigma), 1000 U/ml leukemia inhibitory factor (LIF, Merck)). mESCs were passaged every second day at a density of 4 × 104 cells/cm2 and medium was changed daily. Cells were differentiated by LIF withdrawal in DMEM supplemented with 10% FBS and 0.1 mM β-Mercaptoethanol at a density of 2 × 104 cells/cm2 on fibronectin-coated dishes (Merck, 10 μg/ml).

For the differentiation of mutant cell lines (Fig. 4g), cells were first adapted to 2i + LIF medium (ES cell medium with addition of 3 μM Gsk3 inhibitor CT-99021 (Axon Medchem) and 1 μM Mek inhibitor PD0325901 (Axon Medchem)) for at least five passages before undergoing differentiation via LIF withdrawal (see above). TX1072 XX and XO cells were grown in ES cell medium supplemented with 2i and differentiated by 2i/LIF withdrawal. Hek293T cells were cultured in DMEM supplemented with 10% FBS and passaged every 2 to 3 days.

For the generation of cell lines carrying randomly integrated transgenes using lentiviral transduction, DNA constructs were first packaged into lentiviral particles. For this, 1 × 106 Hek293T cells were seeded into one well of a 6-well plate and transfected the next day with the lentiviral packaging vectors: 1.2 μg pLP1, 0.6 μg pLP2, and 0.4 μg VSVG (Thermo Fisher Scientific), together with 2 μg of the desired construct using Lipofectamine 2000 (Thermo Fisher Scientific). Hek293T supernatant containing the viral particles was harvested after 48 h. 0.2 × 106 mESCs were seeded per 12-well and transduced the next day with 500 μl of viral supernatant and 8 ng/μl polybrene (Sigma). Antibiotic selection was started 2 days after transduction and kept for at least 3 passages.

To generate 1.8-Nanog-mCherry and 1.8-Esrrb-mCherry reporter lines, 1 × 106 1.8 mESCs were transfected with 4 μg of the pUC19-Nanog-mCherry-puro or pUC19-Esrrb-mCherry-puro plasmid (Additional file 9: Table S8) and 1.5 μg of each of the sgRNAs plasmids (PX330-Nanog-sgRNA1/2 and PX330-Esrrb-sgRNA1/2) using 16.5 μl of Lipofectamine 3000 and 22 μl of P3000 (Thermo Fisher Scientific) according to the manufacturer’s recommendations. Cells were selected with puromycin (1 ng/μl, Sigma) for 3 days, starting at day 2 after transfection. The puromycin selection cassette was subsequently excised by transient transfection of a CRE recombinase expression plasmid pCAG-Cre (Addgene 13,775, [94]). Individual clones were expanded and tested for loss of puromycin resistance. mCherry fluorescence was measured via flow cytometry and clones were subsequently genotyped by PCR (Additional file 1: Figure S2B). All PCRs were carried out by using the Hotstart Taq Polymerase (Qiagen), a Tm of 56 °C and 30 cycles (Primer sequences are listed in Additional file 9: Table S8).

In order to generate Klhl13 mutant mESCs, 4 guide RNAs were designed to target a 4.5-kb region around the Klhl13 promoter (2 guide RNAs on each side) with the Alt-R® CRISPR-Cas9 System (IDT), which contains all necessary reagents for the delivery of Cas9-gRNA ribonucleoprotein complexes (RNP) into target cells. Briefly, crRNAs and tracrRNA (gRNA sequences in Additional file 9: Table S8) were mixed in equimolar concentrations and the 4 crRNAs and tracrRNA duplexes were subsequently pooled together. 2.1 μl PBS, 1.2 μl of the tra + cr duplex (100 μM stock), 1.7 μl Cas9 (61 μM stock), and 1 μl electroporation enhancer were pipetted together and incubated for 20 min. In total, 105 cells were nucleofected with the mixture using the CP106 program of the Amaxa 4D-Nucleofector (Lonza) and plated on gelatin-coated 48-well plates. After 48 h, cells were seeded at a density of 10 cells/cm2 into 10-cm plates. Individual clones were picked, expanded, and genotyped for the presence of the promoter deletion. The genotyping strategy is shown in Additional file 1: Figure S4B. For the amplification of the wildtype band, the HotStart Taq Polymerase (Qiagen) was used with an annealing temperature of 51 °C and 35 cycles. For the deletion, the Phusion HiFi Polymerase (NEB) was used with an annealing temperature of 63 °C and 35 cycles (Primer sequences are listed in Additional file 9: Table S8).

For the generation of Dusp9 mutant mESCs, 2 × 106 WT and K13-HET (Clone 1) cells were nucleofected with 5 μg of the PX458-Dusp9_sgRNA1 plasmid (Additional file 9: Table S8) and subsequently plated on gelatin-coated 6-cm plates. The next day, high GFP+ cells were single-cell sorted into a 96-well plate and expanded. Clones were screened for homozygous or heterozygous frameshift deletions via Sanger sequencing and immunoblotting. Heterozygous deletion of several selected clones was further confirmed via NGS. Briefly, a region surrounding the Dusp9 deletion was amplified using the Phusion HiFi Polymerase (NEB) with a total of 30 cycles and an annealing temperature of 65 °C (Primer sequences in Additional file 9: Table S8, OG197/OG198). A second PCR using again the Phusion HiFi Polymerase (NEB) with a total of 14 cycles and an annealing temperature of 65 °C was performed in order to attach the Illumina adaptors and barcodes (Additional file 9: Table S8, OG202/OG210). A dual barcoding strategy was employed, where Illumina barcodes were included in the reverse and custom sample barcodes in the forward primers. Samples containing the same Illumina barcode but different custom sample barcodes were pooled in an equimolar fashion and sequenced on the Illumina Miseq platform PE150. Samples were aligned using Bowtie2 [95] and an index containing sample barcodes and possible deletion sequences based on previously generated Sanger sequencing data, gaining approximately 4000 reads per sample.

The 1.8 XX SP107 (Clone A2) and 1.8 XX K13-HOM SP107 mESC lines stably express PYL1-KRAB-IRES-Blast and ABI-tagBFP-SpdCas9, constituting a two-component CRISPRi system, where dCas9 and the KRAB repressor domain are fused to ABI and PYL1 proteins, respectively, which dimerize upon treatment with abscisic acid (ABA) [96]. The 1.8 XX SP106 mESC line, on the other hand, expresses PYL1-VPR-IRES-Blast instead of PYL1-KRAB-IRES-Blast, together with ABI-tagBFP-SpdCas9, which leads to CRISPR-mediated activation of target genes when recruited to their TSS upon ABA treatment.

The 1.8 XX SP107, 1.8 XX K13-HOM SP107, and 1.8 XX SP106 mESC lines were generated through piggybac transposition. To this end, the puromycin resistance cassettes in the piggybac CRISPRi expression plasmid pSLQ2818 (pPB: CAG-PYL1-KRAB-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9, Addgene 84,241 [96]) and the CRISPRa expression plasmid pSLQ2817 (pPB: CAG-PYL1-VPR-IRES-Puro-WPRE-SV40PA PGK-ABI-tagBFP-SpdCas9, Addgene 84239 [96]) were exchanged for a blasticidin resistance, resulting in plasmid SP107 and SP106, respectively. The SP107 and SP106 plasmids were then, together with the hyperactive transposase (pBroad3_hyPBase_IRES_tagRFP) [97], transfected into the 1.8 XX K13-HOM (Clone 1) and 1.8 XX (Clone 1) mESC lines, respectively, in a 1-to-5 transposase-to-target ratio. RFP-positive cells were sorted 24 h after transfection and cells were selected with blasticidin (5 ng/μl, Roth) for stable construct integration. After expansion, high BFP-positive cells were sorted. For the 1.8 XX SP107 mESCs, a clonal line was generated. Since target gene repression in cell lines stably expressing the SP107 construct transduced with sgRNAs was often observed already without ABA treatment, we could not make use of the inducibility of the system. Instead, 1.8 XX SP107 and 1.8 XX K13-HOM SP107 mESCs were always treated with ABA (100 μM) 5 days before the analysis and effects were compared to NTC sgRNAs. A 5-day ABA treatment (100 μM) was also carried out for the 1.8 XX SP106 mESC line prior to cell harvesting.

Note: The content above has been extracted from a research article, so it may not display correctly.

Also in the Article

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.