sgRNA sequences (Supplementary Table 4 ) were cloned into the pLG1 plasmid (gift from Prof. Jonathan Weissman). Intact pLG1 plasmid that contains sgRNA sequence against EGFP gene was also used as a negative control. sgRNA containing pLG1 vectors along with the pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene, plasmid #60954) were used to generate lentivirus using the Lenti-Pac HIV expression packaging kit (GeneCopoeia). Lentivirus was concentrated using the Lenti-X Concentrator (Takara). mHypoA-POMC/GFP-1 cells were infected with sgRNA and KRAB-dCas9 virus (1:1 ratio) at a multiplicity of infection (MOI) of 0.5. Following 48 hours, RNA was extracted using the RNeasy mini kit (Qiagen) and reverse-transcribed using Superscript III reverse transcriptase (Invitrogen). Gene expression was examined by RT-qPCR using SSO fast EvaGreen supermix (Bio-Rad) was carried out on an Eppendorf Realplex 2. Primers used for the qPCR are shown in Supplementary Table 4 .
Phr sffv krab dcas9 p2a mcherry
Manufactured by Addgene
Sourced in United States
PHR-SFFV-KRAB-dCas9-P2A-mCherry is a plasmid construct containing a catalytically-dead Cas9 (dCas9) fused to the KRAB repressor domain and mCherry fluorescent protein. The construct is driven by the SFFV promoter.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Ssofast evagreen supermix, by Bio-Rad (3 mentions)
Realplex2, by Eppendorf (2 mentions)
Lenti pac hiv expression packaging kit, by GeneCopoeia (2 mentions)
Lenti x concentrator, by Takara Bio (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Polybrene, by Merck Group (2 mentions)
Lentiguide puro, by Addgene (2 mentions)
Lenti mcp lsd1 hygro, by Addgene (2 mentions)
Lenti sgrna ms2 zsgreen1, by Addgene (2 mentions)
Pu6 sgrna ef1alpha puro t2a bfp, by Addgene (2 mentions)
Pspax2, by Addgene (2 mentions)
Pmd2 g, by Addgene (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Sgrna, by Merck Group (1 mentions)
Pklv u6grna bbsi pgkpuro2abfp, by Addgene (1 mentions)
Primestar hs dna polymerase, by Takara Bio (1 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (1 mentions)
Pcdh puro cmyc, by Addgene (1 mentions)
Plko 1 puro vector, by Addgene (1 mentions)
19 protocols using phr sffv krab dcas9 p2a mcherry
1
CRISPR-Cas9 Transcriptional Repression Assay
2
CRISPR-Mediated LTR Silencing in Leukemia
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sgRNAs (Sigma-Aldrich) targeting multiple LTR copies were cloned into lentiviral expression vector pKLV-U6gRNA(BbsI)-PGKpuro2ABFP (Addgene 50946, deposited by K. Yusa). For LTR silencing, OCI-AML3 and K562 cells were first transduced with the lentiviral vector pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene 60954, deposited by Jonathan Weissman), sorted for mCherry on a FACSAria II. Cells expressing mCherry were then subsequently transduced with the lentiviral sgRNA expression vector. Two days later, the cells expressing both mCherry and BFP were sorted and cultured for transcriptional and chromatin analyses.
3
Engineered Lentiviral Vectors for CRISPR/Cas9 Genome Editing
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The sgRNA vector (pLenti-sgRNA-GFP) was cloned by replacing the U6 promoter in pLL3.7 (Addgene) with the human U6 promoter, ccdB cassette, and sgRNA scaffold. The Cas9 expression vector (pLenti-OC-IRES-BSD) has been previously reported [8 (link)]. pcDNA-HBEGF and pcDNA-PSMB5 were cloned by replacing the KRAB-dCas9 element of pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene) with the human HBEGF or PSMB5 coding sequence and 3×FLAG. Vectors expressing cDNA of HBEGF with single-a.a. deletions were constructed via PCR-based site-directed mutagenesis (PfuUltraII Fusion HS DNA Polymerase, STRATAGENE). The primers used for these purposes are listed in Additional file 5 : Table S7. Vectors expressing cDNAs of PSMB5 M104 and V90 substitutions were constructed via PCR-based site-directed mutagenesis (PrimeSTAR HS DNA Polymerase, Takara). Primers used for the construction of M104 and V90 substitution mutants are listed in Additional file 5 : Table S10.
4
Targeted Gene Regulation in Neural Development
5
CRISPR-Cas9 Transcriptional Repression Assay
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sgRNA sequences (Supplementary Table 4 ) were cloned into the pLG1 plasmid (gift from Prof. Jonathan Weissman). Intact pLG1 plasmid that contains sgRNA sequence against EGFP gene was also used as a negative control. sgRNA containing pLG1 vectors along with the pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene, plasmid #60954) were used to generate lentivirus using the Lenti-Pac HIV expression packaging kit (GeneCopoeia). Lentivirus was concentrated using the Lenti-X Concentrator (Takara). mHypoA-POMC/GFP-1 cells were infected with sgRNA and KRAB-dCas9 virus (1:1 ratio) at a multiplicity of infection (MOI) of 0.5. Following 48 hours, RNA was extracted using the RNeasy mini kit (Qiagen) and reverse-transcribed using Superscript III reverse transcriptase (Invitrogen). Gene expression was examined by RT-qPCR using SSO fast EvaGreen supermix (Bio-Rad) was carried out on an Eppendorf Realplex 2. Primers used for the qPCR are shown in Supplementary Table 4 .
6
Lentiviral-mediated Gene Manipulation in Cell Lines
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The shRNAs were cloned into the pLKO.1-puro vector (Addgene, #8453) by following the manufacturer’s instructions. The targeting sequence of each shRNA was listed in Supplementary Data 3 . MSCV-Myc-IRES-RFP (Addgene,#35395) and pCDH-puro-cMyc (Addgene,#46970) were used to expression MYC in the rescue experiments in mouse and human cell lines, respectively. For CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) experiments, gRNAs were cloned into the LentiGuide-Puro (Addgene#52963) vector by using the BsmBI site. pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene#60954) was used to express the dCas9-KRAB fusion protein. Sequences for all the sgRNAs were listed in Supplementary Data 3 .
7
Regulation of CCND1 by Superenhancer Modulation
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First, pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene) and Lenti_MCP-LSD1_Hygro (Addgene) were packaged into a slow-replicating virus. After transducing Huh7 cells with both viral vectors, we selected hygromycin-resistant and mCherry-positive cells to finally obtain the corresponding dCas9-KRAB-LSD1 cells. Next, based on the H3K27ac ChIP-seq and ATAC-seq peaks in Huh7 cells, we found the location of the superenhancer and obtained the DNA sequences of the corresponding ATAC-seq peaks. The sgRNAs used to inhibit the superenhancers were designed with CRISPR-ERA (http://crispr-era.stanford.edu/ ). Annealed double-stranded DNA was inserted into Lenti_sgRNA(MS2)_ZsGreen1 (Addgene) using BsmI (NEB). Then, viruses packaged with the purified recombinant plasmid were used to infect dCas9-KRAB-LSD1 cells in 6-well plates. After screening of ZsGreen-positive cells using flow cytometry, the variation in the mRNA level of CCND1 was obtained using qPCR. Primer sequences are listed in Supplementary Table S1.
8
KRAB-dCas9 Plasmid Construction
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The ZFP3 sequence recognizing CGAGCCCTCAGATGC was synthesized by Genescript (NJ, USA). It was cloned into the plasmid pHR-SFFV-KRAB-dCas9-P2A-mCherry by substitution of KRAB-dCas9 cassette using Gibson assembly (New England Biolabs, Cat#E2611). Later the mCherry was substituted with BFP [50 (link)]. Primers for Gibson assembly are found in S2 Table . pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene plasmid #60954) and pU6-sgRNA-EF1Alpha-puro-T2A-BFP ( Addgene plasmid #60955) were gifts from Jonathan Weissman. Guide RNAs for dCas9 were cloned into the pU6- sgRNA-EF1Alpha-puro-T2A-BFP plasmid.
9
CRISPR-Mediated Silencing of IRF1 Enhancer
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The single guide (sg) RNAs were designed for IRF1 target regions using CHOPCHOP (http://chopchop.cbu.uib.no ). A human genome non-targeting gRNA was used as a negative control. Synthesized oligonucleotides (Integrated DNA Technologies) were annealed and cloned into the Esp3I restriction sites of lentiviral expression vector Lenti_sgRNA_EFS_GFP (Addgene plasmid # 65656 (38 (link))). Sanger sequencing confirmed sgRNAs insertion. The gRNA target sequences are non-targeting, 5’-GTTCCGCGTTACATAACTTA-3’, and IRF1 enhancer targeting, 5’- TCGGCGCGCAGGCACTCAGA-3’.
Lentivirus was produced in HEK293FT cells used to transduce target cells in the presence of 4 µg/mL polybrene (Sigma). For IRF1 Enhancer silencing, hSAECs were first transduced with the lentiviral vector pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene plasmid # 60954 (38 (link))). The vector expresses a fusion protein of mammalian codon-optimized Streptococcus pyogenes dCas9 (DNA 2.0) fused at the N terminus with the Kox1 KRAB domain and two SV40 nuclear localization sequences at the C terminus. mCherry-expressing hSAECs were transduced with the lentiviral sgRNA expression vector. Two days later, the dual mCherry and GFP-expressing cells were sorted and cultured for RSV infection experiments.
Lentivirus was produced in HEK293FT cells used to transduce target cells in the presence of 4 µg/mL polybrene (Sigma). For IRF1 Enhancer silencing, hSAECs were first transduced with the lentiviral vector pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addgene plasmid # 60954 (38 (link))). The vector expresses a fusion protein of mammalian codon-optimized Streptococcus pyogenes dCas9 (DNA 2.0) fused at the N terminus with the Kox1 KRAB domain and two SV40 nuclear localization sequences at the C terminus. mCherry-expressing hSAECs were transduced with the lentiviral sgRNA expression vector. Two days later, the dual mCherry and GFP-expressing cells were sorted and cultured for RSV infection experiments.
10
CRISPRi-mediated Enhancer/Promoter Repression
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HeLa SCC1-mEGFP-AID cells stably expressing dCas9-KRAB for CRISPRi-mediated repression of enhancer or promoter activity were generated by transduction with the lentiviral vector pHR-SFFV-KRAB-dCas9-P2A-mCherry (Gilbert et al., 2014 (link)) (Addgene plasmid #60954) and subsequent sorting for mCherry-positive cells using a FACS Aria III cell sorter (BD Lifesciences). Lentiviral packaging was performed in Lenti-X packaging cells (Takara Bio) transfected with lentiviral transfer plasmids and packaging plasmids helper plasmids pCMV-VSV-G (Addgene plasmid #8454) and pCMVR8.74 (Addgene plasmid #22036) using standard procedures. gRNA oligos (sequences in Table S3 A) were cloned into pETN expression vector (Michlits et al., 2020 (link)) and transfected into packaging cells (293FT, Thermo Fisher, R70007) using Fugene 6 (Promega, E2691). Virus was then harvested and applied to Scc1-EGFP-AID/dCas9-KRAB cells. Infected cells were selected by G418 (GIBCO, 108321-42-2) and used for RT-qPCR.
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