Pl crispr efs trfp

Manufactured by Addgene

The PL-CRISPR.EFS.tRFP is a plasmid construct that allows for the expression of a red fluorescent protein (tRFP) under the control of the constitutive EFS promoter. This plasmid is designed for CRISPR/Cas9 applications.

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Lab products found in correlation

Pspcas9 bb 2a gfp, by Addgene (1 mentions) Pspcas9 bb 2a puro, by Addgene (1 mentions) Lentiguide puro, by Addgene (1 mentions) Plko5 sgrna efs gfp, by Addgene (1 mentions) Plv hu6 sgrna hubc dcas9 krab t2a gfp, by Addgene (1 mentions) Phr sffv dcas9 bfp krab, by Addgene (1 mentions) Pspax2, by Addgene (1 mentions) Pmd2 g, by Addgene (1 mentions) Sh800z cell sorter, by Sony (1 mentions)

4 protocols using pl crispr efs trfp

CRISPR Plasmid Cloning and Validation

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pSpCas9(BB)-2A-Puro (PX459; Catalog # 48139), pSpCas9(BB)-2A-GFP (PX458; Catalog # 48318), lentiGuide-Puro (Catalog # 52963), pL-CRISPR.EFS.tRFP (Catalog # 57819), pLKO5.sgRNA.EFS.GFP (Catalog # 57822), pLV hU6-sgRNA hUbC-dCas9-KRAB-T2a-GFP (Catalog # 71237), pHR-SFFV-dCas9-BFP-KRAB (Catalog # 46911), pMD2.G (Catalog # 12259) and psPAX2 (Catalog # 12260) were obtained from Addgene. sgRNAs were cloned in appropriate vectors according to manufacturer instructions. Top and bottom oligonucleotides constituting sgRNAs were cloned into appropriate plasmids optimized for mammalian expression. Cloned plasmids were subsequently sequenced for verification of correct sgRNA sequence and orientation.

Das S, & Chadwick B.P. (2021). CRISPR mediated targeting of DUX4 distal regulatory element represses DUX4 target genes dysregulated in Facioscapulohumeral muscular dystrophy. Scientific Reports, 11, 12598.

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CRISPR SgRNA Library for Targeted Genes

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A sgRNA library was designed to cover six protein classes including kinases, nuclear receptors, cell surface proteins (Bausch-Fluck et al, 2015 (link)), epigenetic factors, transcription factors, and uncharacterized genes. Each gene was targeted with 3–12 different sgRNAs, selected to specifically bind to either the first exon, an early splicing site, or the functional domain of the protein. All sgRNAs were chosen to fulfill the criteria defined by Doench et al (2016) (link). The complete library consisted of 10,722 sgRNAs targeting 1,572 genes. 632 sgRNAs were included to target 45 essential and 47 non-essential control genes. Oligonucleotides with sgRNA sequences were ordered as arrayed synthesis from CustomArray Inc. and PCR amplified (primer, forward: 5′-GATATTGCAACGTCTCACACC-3′, reverse: 5′-GTCGCGTACGTCTCGAAAC-3′). The resulting PCR product was cloned into the lentiviral vector pL.CRISPR.EFS.tRFP (57819; Addgene) using the Esp3I restriction site. The vector contained a modified tracr sequence (5′-GTTTAAGAGCTATGCTGGAAACAGCATAGCAAGTTTAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-3′) as previously described (Chen et al, 2013 (link)).

Döring M., Brux M., Paszkowski-Rogacz M., Guillem-Gloria P.M., Buchholz F., Pisabarro M.T, & Theis M. (2024). Nucleolar protein TAAP1/C22orf46 confers pro-survival signaling in non-small cell lung cancer. Life Science Alliance, 7(4), e202302257.

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CRISPR-Cas9 Targeted Knockout Cell Lines

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Targeted knockout cell lines were generated using the CRISPR–Cas9 system either by transient transfection or transduction. For the latter approach, 293T cells were transfected with the plasmid pL-CRISPR.EFS.tRFP (Addgene, 57819) containing the sgRNA for a gene of interest, and the lentiviral packaging plasmids pMDLg/pRRE, pRSV-Rev and VSV.G. Cells were transduced following standard procedures and RFP-positive cells were sorted on a Sony SH800Z cell sorter. Alternatively, cells were transiently transfected using pX330 plasmids containing the sgRNA of interest along with vectors encoding either puromycin or blasticidin resistance to allow transient selection of transfected cells. For HAP1, 293T and HeLa cells, clonal progeny was generated and verified by PCR and Sanger sequencing.

Fessler E., Eckl E.M., Schmitt S., Mancilla I.A., Meyer-Bender M.F., Hanf M., Philippou-Massier J., Krebs S., Zischka H, & Jae L.T. (2020). A pathway coordinated by DELE1 relays mitochondrial stress to the cytosol. Nature, 579(7799), 433-437.

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Generation of CRISPR/Cas9 Knockout Cell Lines

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The pLCP2B plasmid was generated by removing Cas9-P2A-tRFP from pL-CRISPR.EFS.tRFP (Addgene, no. 57819) and replaced with Cas9-P2A-Blast. The lentiCRISPR-v2 vector with puromycin was purchased from Addgene (no. 52961). The sgRNA oligonucleotides described below were engineered into vectors using the standard CRISPR/Cas9 knockout protocol. M3 sgCon cells were pooled from 6 single-cell clones, and M3 sgSUZ12 cells were pooled from 9 single-cell clones. Both SKP605 sgCon cells and sgEed cells were pooled from 4 single-cell clones. See the Supplemental Methods for details on the methods used and sgRNA and sequences.

Yan J., Chen Y., Patel A.J., Warda S., Lee C.J., Nixon B.G., Wong E.W., Miranda-Román M.A., Yang N., Wang Y., Pachai M.R., Sher J., Giff E., Tang F., Khurana E., Singer S., Liu Y., Galbo PM J.r., Maag J.L., Koche R.P., Zheng D., Antonescu C.R., Deng L., Li M.O., Chen Y, & Chi P. (2022). Tumor-intrinsic PRC2 inactivation drives a context-dependent immune-desert microenvironment and is sensitized by immunogenic viruses. The Journal of Clinical Investigation, 132(17), e153437.

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