Lenticas9 egfp

Manufactured by Addgene

Sourced in United States

LentiCas9-EGFP is a lentiviral vector that expresses Cas9 and enhanced green fluorescent protein (EGFP). Cas9 is a programmable RNA-guided endonuclease that can be used for genome editing applications. The EGFP expression allows for the identification of transduced cells.

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8 protocols using lenticas9 egfp

CRISPR-Mediated CD226 Gene Knockout

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An established LCL was transduced with the enhanced green fluorescent protein (EGFP) expression construct lentiCas9-EGFP (Addgene catalog no. 63592) and then subsequently transduced with one of three short guide RNAs (sgRNAs) expressed on the lentiguide-Puro vector (Addgene catalog no. 52963) to target the CD226 gene. Transduced cells were selected with puromycin and screened for efficiency of Cas9 cleavage by the Surveyor nuclease assay (37 (link)). Transduced cell lines were then assayed for CD226 mRNA and surface expression, of which two sgRNAs (sgCD226-1 and CD226-2) showed significantly reduced CD226 expression. Genetic lesions induced by Cas9 cleavage and error-prone DNA repair were sequenced by TOPO TA cloning (Invitrogen catalog no. 45003).

Grossman L., Chang C., Dai J., Nikitin P.A., Jima D.D., Dave S.S, & Luftig M.A. (2017). Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines. mSphere, 2(6), e00305-17.

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Generation of BFL-1 CRISPR/Cas9 Mutant LCLs

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The BFL-1 CRISPR/Cas9 mutant LCL was generated by the Duke Functional Genomics Shared Resource. Cas9-expressing LCLs (lentiCas9-EGFP; Addgene #63592) were transduced with one of three short-guide RNAs (sgRNAs) that were specific to BFL-1 exon 1 and grown out under puromycin selection (lentiGuide-Puro; Addgene #52963). Each sgRNA was screened for efficiency of Cas9 cleavage by Surveyor nuclease assay and for reduced BFL-1 mRNA expression. LCLs transduced with the sgRNA resulting in both Cas9 cleavage of the target site and the most reduced mRNA expression were then serially diluted to obtain a clonal population. Clonality was ascertained by TOPO TA cloning (Invitrogen, Cat #45003).

Price A.M., Dai J., Bazot Q., Patel L., Nikitin P.A., Djavadian R., Winter P.S., Salinas C.A., Barry A.P., Wood K.C., Johannsen E.C., Letai A., Allday M.J, & Luftig M.A. (2017). Epstein-Barr virus ensures B cell survival by uniquely modulating apoptosis at early and late times after infection. eLife, 6, e22509.

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Lenti-HA-RFP and Lenti-OVA Constructs

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Lenti-HA-RFP was generated by PCR amplification of the Puerto Rico influenza A strain 8 haemagglutinin sequence (a gift from T. Griffith) followed by ligation into the lentiCas9-EGFP (Addgene 63592) backbone by restriction enzyme digestion to remove the Cas9 open reading frame and Gibson assembly. Monomeric RFP (mRFP) was subsequently inserted and replaced EGFP by restriction enzyme digestion and Gibson assembly. Lenti-OVA was generated internally, with the OVA sequence^{31 (link)} cloned into a pLVX-EF1a-IRES-neo backbone and Flag-tagged in the N terminus (pLVX-EF1a-IRES-neo: OVA, Flag N-term). Lenti-Cas9–2A-Blast plasmid (Addgene 73310) was used to make Cas9 stably expressing cell lines. The lentiviral firefly luciferase construct (PGK-GFP-IRES-LUCIFERASE) was used to make cell lines that stably express luciferase. The modified lentiCRISPRv2 (ref. ^{12 (link)}) and pLCKO2 plasmids^{32 (link)} (Addgene 125518) were used for expression of individual gRNAs in native or Cas9-expressing cell lines, respectively.

Lawson K.A., Sousa C.M., Zhang X., Kim E., Akthar R., Caumanns J.J., Yao Y., Mikolajewicz N., Ross C., Brown K.R., Zid A.A., Fan Z.P., Hui S., Krall J.A., Simons D.M., Slater C.J., De Jesus V., Tang L., Singh R., Goldford J.E., Martin S., Huang Q., Francis E.A., Habsid A., Climie R., Tieu D., Wei J., Li R., Tong A.H., Aregger M., Chan K.S., Han H., Wang X., Mero P., Brumell J.H., Finelli A., Ailles L., Bader G., Smolen G.A., Kingsbury G.A., Hart T., Kung C, & Moffat J. (2020). Functional genomic landscape of cancer-intrinsic evasion of killing by T cells. Nature, 586(7827), 120-126.

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Protocol for Targeted Gene Knock-in and CRISPR-mediated Mutagenesis

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Px330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene #42230)^{34 (link)}, pKLV-U6gRNA(BbsI)-PGKPuro2ABFP (Addgene #50946)^{35 (link)} and pKLV-U6gRNA(BbsI)-PGKHyg2AEGFP (adapted from Addgene #50946)^{35 (link)} were used for sgRNA delivery. The HDR template for targeted gene knock-in was cloned into a truncated pcDNA3.1(−) (Thermo #V79520). The template was generated by step-wise insertion of T2A-mCherry, PCR amplified from pU6-sgCXCR4-2 (Addgene #46917)^{20 (link)}, PGK-HygR, PCR amplified from pRetroX-Tight-Hygro (Clontech #631034) and the two homology arms that were PCR amplified from genomic DNA by PCR. pHR-SFFV-dCas9-BFP-KRAB (Addgene #46911)^{20 (link)} and tandem HIF2A-targeting sgRNA constructs, cloned as previously described^{10 (link)}, were used for HIF2A CRISPRi. Lenti-Cas9-EGFP (adapted from Addgene #52962)^{36 (link)} was used for HIF2A mutagenesis. pLVX-puro (Clontech #632164) was used to express HA-VHL (Addgene #19234)^{37 (link)}. Primer and sgRNA sequences used in this study are listed in Supplementary Table 3.

Zaini M.N., Patel S.A., Syafruddin S.E., Rodrigues P, & Vanharanta S. (2018). Endogenous HIF2A reporter systems for high-throughput functional screening. Scientific Reports, 8, 12063.

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Engineered CRISPR Plasmid for Efficient Genome Editing

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PX330A_D10A-1×2 (Addgene #58772) was modified by adding a ClaI site in front of hU6 promoter, termed as new pX330A_D10A-1×2. gRNAs were designed using Feng Zhang lab’s online tool (http://crispr.mit.edu/). After annealing of gRNA oligos, the gRNAs duplexes were cloned to new pX330A_D10A-1×2 and pX330S-2 (Addgene #58778) using BbsI (BpiI) (Supplementary Table 5). Through Golden gate assembly using Eco31I, a new pX330A_D10A-1×2, which contains gRNA cassette containing two hU6 promoter and two gRNAs was made. Using Cla1 and Kpn1, the gRNA cassette from this vector was inserted to new lentiCRISPR v2 (modified from lentiCRISPR v2, Addgene #52961, by adding new cloning site containing Cla1 and Kpn1). Finally, the DNA element containing the gRNA cassette of this new lentiCRISPR v2 was replaced with lentiCas9-EGFP (Addgene #63592) using Not1 and Nhe1. Plasmids and gRNA sequences are listed in Supplementary Table 6.

Fu Y., Wang J., Panangipalli G., Ulrich B.J., Koh B., Xu C., Kharwadkar R., Chu X., Wang Y., Gao H., Wu W., Sun J., Tepper R.S., Zhou B., Janga S.C., Yang K, & Kaplan M.H. (2020). STAT5 promotes accessibility and is required for BATF-mediated plasticity at the Il9 locus. Nature Communications, 11, 4882.

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Generation and Validation of CRISPR-Cas9 Knockout Cells

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LentiCas9-EGFP (Donated by Dr. Phil Sharp & Dr. Feng Zhang) and pCW-Cas9 (Donated by Dr. Eric Lander & Dr. David Sabatini) were purchased from Addgene. All Lenti-guided RNA (gRNA)-BFP vectors were purchased from the Functional Genomics Shared Resource at the University of Colorado Cancer Center. MOLM-14 cells were transduced with LentiCas9-EGFP and Lenti-gRNA-BFP viruses sequentially and GFP⁺/BFP⁺ cells were sorted on day 7 for expansion as pools and single clones. Isolation, subcloning and sequencing of edited genomic DNA was performed from samples on day 26. Human leukemia cell lines transduced with pCW-Cas9 were selected and maintained in culture medium with 2.5 ug/mL puromycin. Two days after transducing with gRNA lentivirus, cells were treated with 1 ug/mL doxycycline (except KASUMI-1 which was treated with 0.25 ug/mL doxycycline) to induce Cas9 expression. CD38 gRNA was used as the positive control and the targeted mutation of CD38 gene was detected through disappearance of surface CD38 by flow cytometry.

Chen Y., Anastassiadis K., Kranz A., Stewart A.F., Arndt K., Waskow C., Yokoyama A., Jones K., Neff T., Lee Y, & Ernst P. (2017). MLL2, not MLL1, Plays a Major Role in Sustaining MLL-rearranged Acute Myeloid Leukemia. Cancer cell, 31(6), 755-770.e6.

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Engineered CRISPR/Cas9 Lentiviral Vector

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PX330A_D10A-1 × 2 (Addgene #58772) was modified by adding ClaI site in front of hU6 promoter, termed as “new pX330A_D10A-1 × 2”. gRNAs were designed using the Zhang lab’s online tool (http://crispr.mit.edu/). After annealing of gRNA oligos, the gRNA duplexes were cloned to modified pX330A_D10A-1 × 2 and pX330S-2 (Addgene #58778) using BbsI (BpiI) (Supplementary Table 2). Through Golden gate assembly using Eco31I, the modified pX330A_D10A-1 × 2, which contains gRNA cassette containing two hU6 promoter and two gRNAs was generated. By using Cla1 and Kpn1, the gRNA cassette from this vector was inserted to new lentiCRISPR v2 (modified from lentiCRISPR v2, Addgene #52961, by adding new cloning site containing Cla1 and Kpn1) using Cla1 and Kpn1. Finally, a DNA element containing gRNA cassette of this new lentiCRISPR v2 was replaced with lentiCas9-EGFP (Addgene #63592) using Not1 and Nhe1. Plasmids and gRNA sequences are listed in Supplementary Table 2 and 3.

Koh B., Abdul Qayum A., Srivastava R., Fu Y., Ulrich B.J., Janga S.C, & Kaplan M.H. (2018). A conserved enhancer regulates Il9 expression in multiple lineages. Nature Communications, 9, 4803.

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Immunohistochemistry and Lentiviral Plasmids

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Antibodies against PARP, cleaved PARP, Bcl-2, Bcl-XL, Bax, β-actin, Cyclin D1, Cyclin D3, Ki67, and CD31 were bought from Cell Signaling (Danvers, MA, United States). Mouse LMO7 antibody was purchased from Santa Cruz (California, CA, United States). All immunohistochemistry (IHC) reagents including ImmPRESS^TM HRP anti-rabbit IgG (Peroxidase) (Cat#MP-7401), ImmPACT DAB peroxidase (HRP) substrate (Cat#SK-4105), and hematoxylin (Cat#H-3404) were purchased from Vector Laboratories (Burlingame, CA, United States). Plasmids pLL3.7 and LentiCas9-EGFP (Cat#63592) were bought from Addgene (Cambridge, MA, United States).

Liu X., Yuan H., Zhou J., Wang Q., Qi X., Bernal C., Avella D., Kaifi J.T., Kimchi E.T., Timothy P., Cheng K., Miao Y., Jiang K, & Li G. (2021). LMO7 as an Unrecognized Factor Promoting Pancreatic Cancer Progression and Metastasis. Frontiers in Cell and Developmental Biology, 9, 647387.

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