Lenti sgrna ms2 puro

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 the CRISPR/dCAS9 gene editing system to activate endogenous Nrf1 expression according to the published protocol.
²⁸ (link) Briefly, sgRNA targeting Nrf1 was cloned into Lenti‐sgRNA (MS2)_puro (Addgene, #73795). The dCas9‐VP64 (Addgene, #61425) and MS2‐P65‐HSF1 (Addgene, #61426) were introduced simultaneously into PSCs to activate endogenous Nrf1 expression. To construct NRF1 OE plasmid, Nrf1 cDNA was amplified from mouse PSCs and cloned into SalI and NheI sites of a modified pInducer20 (p20) vector (Addgene, #44012). The correctly inserted sgRNA sequence and Nrf1 cDNA in the expression vectors were confirmed by Sanger sequencing. The sequences of sgRNA for Nrf1 activation and primers for Nrf1 cDNA cloning are listed in Table S4.