Lenti sgrna ms2 zsgreen1

Manufactured by Addgene

Lenti_sgRNA(MS2)_ZsGreen1 is a lentiviral vector designed for CRISPR/Cas9-mediated gene editing. The vector contains an MS2-tagged single guide RNA (sgRNA) and expresses the ZsGreen1 fluorescent reporter protein.

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Lab products found in correlation

Phr sffv krab dcas9 p2a mcherry, by Addgene (2 mentions) Lenti mcp lsd1 hygro, by Addgene (2 mentions) Lentiguide puro, by Addgene (1 mentions) Seamless cloning and assembly kit, by Transgene (1 mentions)

2 protocols using lenti sgrna ms2 zsgreen1

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.

Liu Z., Ye Y., Liu Y., Liu Y., Chen H., Shen M., Wang Z., Huang S., Han L., Chen Z, & He X. (2022). RNA Helicase DHX37 Facilitates Liver Cancer Progression by Cooperating with PLRG1 to Drive Superenhancer-Mediated Transcription of Cyclin D1. Cancer Research, 82(10), 1937-1952.

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CRISPR-based Transcriptional Regulation of EHF and ELF3

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The open reading frames (ORFs) of EHF and ELF3 were amplified from HEK293T cell cDNA and cloned into pCDH‐CMV‐3X‐Flag‐Puro (SBI, Palo Alto, CA, USA) using Seamless Cloning and Assembly Kit (Transgen Biotech, Beijing, China). For enCRISPRi assay, pHR‐SFFV‐KRAB‐dCas9‐P2A‐mCherry and Lenti_MCP‐LSD1_Hygro plasmids were purchased from Addgene (Watertown, USA). sgRNA sequences targeting SEs of KLF5 were designed by CRISPR‐ERA (http://crispr‐era.stanford.edu/) and inserted into the Lenti_sgRNA(MS2)_ZsGreen1 (Addgene) using BsmbI (NEB). sgRNA sequences targeting KLF5 protein‐coding regions were designed by CRISPR‐ERA (http://crispr‐era.stanford.edu/) and inserted into Lenti‐guide‐puro (Addgene) using BsmbI. The RAD51 promoter sequence containing the KLF5 binding site was amplified from HEK293T cell cDNA and cloned into PGL3‐enhancer (Progema, Madison, USA). The sgRNA sequences are provided in Table S7 (Supporting Information).

Wu Y., Chen S., Shao Y., Su Y., Li Q., Wu J., Zhu J., Wen H., Huang Y., Zheng Z., Chen X., Ju X., Huang S., Wu X, & Hu Z. (2023). KLF5 Promotes Tumor Progression and Parp Inhibitor Resistance in Ovarian Cancer. Advanced Science, 10(31), 2304638.

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