Fgh1tutg

For lentiviral packaging, HEK293T cells were seeded in a 10 cm dish 1 day before transfection. The indicated viral plasmids Lentidcas9-vp64-blast (Addgene #61425) or FgH1tUTG (Addgene #70183) were co-transfected with lentivirus packaging plasmids pMD2.G (Addgene #12259) and psPAX2 (Addgene #12260) in a 4:2:3 ratio using PEI MAX (molecular weight 40,000) (Polysciences, Inc., USA). The next day after transfection, media were changed to Opti-MEM® I Reduced Serum Media (Thermo Fisher Scientific). At 48 h after transfection, virus-containing media were collected by Amicon Ultra-15 Centrifugal Filter Units 100 kDa (Millipore), passed through a 0.45 µm polyethersulfone filter (Millipore), aliquoted and stored at −80 °C before infection or titration. mESCs were seeded 1 day before transduction. The next day, the medium was changed to fresh and protamine sulfate 50 μg/ml (Sigma) and Lentidcas9-vp64-blast or FgH1tUTG were added. Infected green fluorescent protein-positive cells mESCs were sorted in BD FACSAria™ III. FgH1tUTG-transduced cells were infected with Lentidcas9-vp64-blast and selected by 5 µg/ml of blasticidin (Sigma) for 1 week. Oligonucleotides were designed using the online tools http://sam.genome-engineering.org/database and https://www.nature.com/articles/nbt.2647: F-TCCCGAGGCGGGGCGGGGCTTAGTT; R-AAACAACTAAGCCCCGCCCCGCCTC; and cloned into FgH1tUTG vector.

To generate MSMO- or FAXDC2-KO HPAF-II cells, MSMO1 sgRNA (sg: 5′-CACCGCAGAGACATGGGAAAACCAA-3′) or FAXDC2 sgRNAs (sg3: 5′-TCTTGTTCTACTATTCACAC-3′; sg4: 5′-TGGGGAAAGATATCATGCAC-3′) were cloned into FgH1tUTCyan (85551, Addgene) and FgH1tUTG (70183, Addgene) plasmids, respectively.
Lentiviral particles were produced by transient transfection of 293T cells grown in 60 mm Petri dishes with 10 μg of vector DNA along with the packaging constructs pMD2.G (12259, Addgene) and psPAX2 (12260, Addgene) using Lipofectamine 2000. Virus-containing supernatants were collected at 48–72 hours after transfection and passed through a 0.45 mm filter. HPAF-II cells stably expressing FUCas9Cherry (70182, Addgene) were transduced with virus-containing supernatant with 10 ng/mL Polybrene. Genome editing was validated by PCR amplification and sequencing.

For deleting a peak of SE by CRISPR/Cas9 editing, a pair of gRNAs franking the peak were designed using http://crispr.mit.edu/. The pair of gRNAs (Supplementary Table 4) were then sequentially inserted into BsaI and BbsI restriction sites of px333 vector (Addgene 64073), which encodes spCas9 and 2 sgRNA cassettes. For deleting the SE peak with a lentiviral vector, FgH1tUTG (Addgene #70183) was used. The sequence of hU6 promoter-sgRNA-hU6 promoter-sgRNA was amplified by PCR and then cloned into PacI digested FgH1tUTG (to remove the H1t promoter and gRNA scaffold). shRNAs were cloned into pLKO.1 vector (Addgene 8453) or Tet-pLKO-puro (Addgene 21915) digested by AgeI and EcoRI. FOXC1 CDS was cloned into the CD532A-1 vector by Genewiz company.

To knock out TCOF1 and KIT, Crispr/Cas9 knockout system was used. FUCas9Cherry (#70182), lentiV_Cas9_puro (#108100) and FgH1tUTG (#70183) were ordered from Addgene. Guide RNAs (gRNAs) was designed using online tool www.crisprscan.org/ and http://crispr.mit.edu/. gRNA oligos (Table S1) with sticky end were synthesised by IDT company. Then gRNAs were cloned into BsmBI restriction site of FgH1tUTG vector. To overexpress endogenous TCOF1, CRISPR/Cas9 Synergistic Activation Mediator (SAM) system was used; vectors of lenti-dcas-vp64_blast (#61425), lenti-sgRNA(MS2)_puro (#73795) and lentiMPH v2 (89308) were purchased from Addgene. To overexpress exogenous HA-TCOF1, CDS of TCOF1 with HA tag at N-terminal was synthesised and cloned into CD532A-1 vector by GENEWIZ. To overexpress exogenous FZD8-HA, KIT-HA, CDS of FZD8 and KIT with HA tag at C-terminal were synthesised and cloned into CD532A-1 vector by GENEWIZ. To delete super-enhancer peak, http://crispr.mit.edu/ was used to design a pair of gRNA franking the peak. The gRNA pairs were then inserted into BbsI and BsaI restriction sites of px333 vector (Addgene 64073). PCR was employed to amplify hU6 promoter-sgRNA-hU6 promoter-sgRNA. The sequence was then cloned into the PacI-digested lentiviral vector FgH1tUTG.

To knockout ANLN, Crispr/Cas9 inducible knockout system was used. FUCas9Cherry (#70182) and FgH1tUTG (#70183) were ordered from Addgene. Guide RNAs (gRNAs) were designed using online tools http://crisper.mit.edu/ and http://crisprscan.org. gRNAs oligos (Supplementary Table S1) with sticky end were synthesized by IDT company. The gRNAs were cloned into restriction BsmBI restriction sites of FgH1tUTG vector. For overexpression of exogenous BMP2-HA, CDS of BMP2 with HA tag at C-terminal was synthesized and cloned into vector CD532A-1 by GENEWIZ. To overexpress HA-TWIST1, CDS of TWIST1 with HA-tag at N-terminal were synthesized and cloned into vector CD532A-1 by GENEWIZ. For peak deletion of super-enhancer, a pair of gRNAs flanking the peak were designed using http://crisper.mit.edu/ (Supplementary Table S1). The pair of gRNAs were then inserted sequentially into BsaI and BbsI restriction sites of pX333 vector (Addgene #64073) that encodes spCas9 and two gRNA cassettes.