Lenti dcas vp64 blast

MCF-7 cells were first infected with lenti MS2-P65-HSF1_Hygro (Addgene Plasmid #61426) and lenti dCAS-VP64_Blast (Addgene Plasmid #61425) and then selected with hygromycin and blasticidin. Next, SAM library was introduced into these cells by infection, followed by zeocin selection. The cells were cultured in E2 free medium for 28 days during which the medium was replaced with fresh E2 free medium every other 2 days. After that, the cells were harvested for lncRNA profiling.

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.

We used human codon-optimized spCas9 [p3s-Cas9HC (Addgene plasmids #43945)], LbCpf1 [pY016 (Addgene plasmids #69988)], and AsCpf1 [pY010 (Addgene plasmids #69982)]. Plasmid DNAs for orthogonal gene manipulation, lenti dCAS-VP64_Blast (Addgene plasmid #61425) and lenti MS2-p65-HSF1_Hygro (Addgene plasmid #61426) were gifts from Feng Zhang. pU6-As-crRNA (Addgene plasmid #78956) and pU6-Lb-crRNA (Addgene plasmid #78957) were used for cloning the gRNA of LbCpf1 and AsCpf1. To construct fgRNA-cloning vector, 5′-scaffold sequences of LbCpf1 (5′-AATTTCTACTAAGTGTAGAT-3′) and AsCpf1 (5′-TAATTTCTACTCTTGTAGAT-3′) were inserted behind the U6 promoter sequences of the Cas9 gRNA-cloning vector. The target sequences of each gRNA are listed in Supplementary Table 1.

The Lenti-idCas9-KRAB-neo and Lenti-idCas9-VP64-neo plasmids were generated from Lenti-iCas9-neo (Addgene, plasmid # 85,400), TRE-KRAB-dCas9-IRES-GFP (Addgene, plasmid # 85,556) and lenti dCAS-VP64_Blast (Addgene, plasmid # 61,425). The lentiMPH v2-EGFP plasmid was generated by replacing hygromycin with EGFP from lentiMPH v2 (Addgene, plasmid # 89,308). CROPseq-i-sgRNA-BFP and CROPseq-a-sgRNA-BFP backbones were generated from CROPseq-Guide-Puro (Addgene, plasmid # 86,708). CROPseq-i-NC-BFP and CROPseq-i-KRAS-BFP plasmids were generated from CROPseq-i-sgRNA-BFP backbone with i-NC or i-KRAS, respectively. CROPseq-a-NC-BFP. CROPseq-a-KRAS-1-BFP and CROPseq-a-KRAS-2-BFP plasmids were generated from CROPseq-a-sgRNA-BFP backbone with a-NC, a-KRAS-1, or a-KRAS-2, respectively. The sgRNA sequences are listed in Supplementary Table 8. Lenti-iCas9-neo was a gift from Qin Yan, TRE-KRAB-dCas9-IRES-GFP was a gift from Eric Lander, lenti dCAS-VP64_Blast and lentiMPH v2 were gifts from Feng Zhang, and CROPseq-Guide-Puro was a gift from Christoph Bock [35 (link)–39 (link)].

vWF was overexpressed in HLE cells using CRISPR SAM involving three vectors, Lenti dCAS‐VP64_Blast (#61 425, Addgene), LentiMPH v2 (#89 308, Addgene), and sgRNA(MS2)_zeo backbone (#61 427, Addgene) that carries sgRNA of vWF. sgRNA of vWF was subcloned into sgRNA(MS2)_zeo backbone via BsmBI site. The sequences of oligos of sgRNA are listed in Table S3 (Supporting Information). The successful subcloning of vWF sgRNA sequences into sgRNA(MS2)_zeo backbone was confirmed by DNA sequencing. Lentiviral plasmids were transfected into HEK293FT cells using Lenti‐Pac HIV Expression Packaging Kit (#LT001, GeneCopoeia) according to manufacturer's protocol. The viral supernatant was collected, centrifuged, and filtered before using for transduction of HCC cells. Polybrene (8 µg mL⁻¹) (Sigma–Aldrich) was added as a transduction enhancer. HCC cells were selected by blasticidin (#A1113903, Thermo Fisher Scientific), puromycin (#A1113803, Thermo Fisher Scientific), and zeocin (#ant‐zn‐1, InvivoGen) according to the vector information. vWF‐SAM1, vWF‐SAM2 and respective Control clones were established.