Px458

Manufactured by Addgene

Sourced in United States

PX458 is a plasmid that contains a CRISPR/Cas9 system. It includes a Cas9 expression cassette and a cloning site for single guide RNA (sgRNA) insertion. This plasmid can be used for genome editing applications.

Automatically generated - may contain errors

Lab products found in correlation

Lipofectamine 2000, by Thermo Fisher Scientific (9 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (9 mentions) Px459, by Addgene (6 mentions) Facsaria, by BD (5 mentions) Px330, by Addgene (4 mentions) Pspcas9 bb 2a gfp, by Addgene (3 mentions) Amaxa 4d nucleofector x unit, by Lonza (3 mentions) Facsaria 3, by BD (3 mentions) Megashortscript t7 transcription kit, by Thermo Fisher Scientific (3 mentions) Pcdh ef1 mcs neo and piggybac, by New England Biolabs (2 mentions) Ppb cag 3xflag empty pgk hph, by Addgene (2 mentions) Dneasy blood and tissue kit, by Qiagen (2 mentions) Xfect transfection reagent, by Takara Bio (2 mentions) Lenticrisprv2 mcherry, by Addgene (2 mentions) Px458, by Takara Bio (2 mentions) Px458, by Mirus Bio (2 mentions) Lenticrispr v2, by Addgene (2 mentions) Jetprime, by Polyplus Transfection (2 mentions) Px333, by Addgene (2 mentions) X tremegene 9, by Roche (2 mentions)

Spelling variants of this product

PSpCas9(BB)-2A-GFP (PX458) vector (39 citations) PSpCas9(BB)-2A-GFP (PX458) plasmid (34 citations) PX458-GFP (5 citations) 2A-EGFP pSpCas9 (BB)-2A-GFP (PX458) (2 citations) PX458 CRISPR/Cas9-GFP vector (2 citations) PX458-sgRNA_Ago1_5/6 (2 citations) Px458 CRISPR/Cas9 vector (2 citations) PX458-GFP vector (2 citations) PX458 CRISPR (2 citations) PSpCas9(BB)‐2A‐GFP (PX458; 48138; (2 citations)

110 protocols using px458

Targeted Glycosyltransferase Inactivation by CRISPR

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Example 8

Inactivation of Glycosyltransferase Genes by Multi-Exon Dual CRISPR/Cas9 Gene Editing.

Gene inactivation is ensured by removal of exons encoding the cytoplasmic tail/transmembrane and parts of the stem encoded sequences of the desired gene needed to be inactivated. Target region deletion is mediated by use of a pairs of CRISPR/Cas9-gRNAs targeting the flanking regions encoding Mgat4a signal sequence, transmembrane anchoring and stem-regions. CHO Mgat4a is targeted using CHO Mgat4a CRISPR/Cas9 gRNA's Mgat4aEx1gRNA (5′-GGTATACCACATGGCAAAATGGG-3′) specific for exon1 and Mgat4aEx2gRNA (5′-GTCCAACAGTTTCGCCGTGCAGG-3′) specific for exon2. Both gRNA's were cloned into the BbsI (NEB, USA) gRNA target site of px458 (Addgene, USA) encoding GFP tagged S. pyogenes Cas9 and U6 promoter driven gRNA casette, generating px458-CRISPR-Mgat4aex1gRNA and px458-CRISPR-Mgat4aex2gRNA. Precise gene editing by nucleofection using 2 ug of these CRISPR/Cas9-Mgat4a editing tools and target selection validation was performed in CHO-GS cells as described in Examples 3 and 7.

US10858671B2. N-glycosylation (2020-12-08). University of Copenhagen [DK], DANMARKS TEKNISKE UNIVERSITET [DK]. Inventors: Henrik Clausen [DK], Zhang Yang [DK], Adnan Fevzi Halim [SE], Eric Bennett [DK], Carsten Behrens [DK], Malene Bech Vester-Christensen [DK], Shamim Herbert Rahman [DK].

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Inactivating Glycosyltransferase Genes via CRISPR

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Example 7

Inactivation of Glycosyltransferase Genes by Multi-Exon Dual CRISPR/Cas9 Gene Editing.

Gene inactivation is ensured by removal of exons encoding the cytoplasmic tail/transmembrane and parts of the stem encoded sequences of the desired gene needed to be inactivated. Target region deletion is mediated by use of a pairs of CRISPR/Cas9-gRNAs targeting the flanking regions encoding Mgat4a signal sequence, transmembrane anchoring and stem-regions. CHO Mgat4a is targeted using CHO Mgat4a CRISPR/Cas9 gRNA's Mgat4aEx1gRNA (5′-GGTATACCACATGGCAAAATGGG-3′) (SEQ ID NO:58) specific for exon1 andMgat4aEx2gRNA (5′-GTCCAACAGTTTCGCCGTGCAGG-3′) (SEQ ID NO:59) specific for exon2. Both gRNA's were cloned into the BbsI (NEB, USA) gRNA target site of px458 (Addgene, USA) encoding GFP tagged S. pyogenes Cas9 and U6 promoter driven gRNA casette, generating px458-CRISPR-Mgat4aex1gRNA and px458-CRISPR-Mgat4aex2gRNA. Precise gene editing by nucleofection using 2 ug of these CRISPR/Cas9-Mgat4a editing tools and target selection validation was performed in CHO-GS cells as described in Examples 2 and 6.

US10889845B2. Production of N-glycoproteins for enzyme assisted glycomodification (2021-01-12). UNIVERSITY OF COPENHAGEN [DK], DANMARKS TEKNISKE UNIVERSITET [DK]. Inventors: Shamim Herbert Rahman [DK], Carsten Behrens [DK], Malene Bech Vester-Christensen [DK], Henrik Clausen [DK], Zhang Yang [DK], Adnan Fevzi Halim [SE], Eric Bennett [DK].

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AID-Mediated Homozygous Tagging of SPT6

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The AID sequence was designed as published earlier (Muhar et al., 2018 (link)). For homozygous tagging of SPT6, pJET-CAID-Blast-entry-vector was constructed using the AID sequence. Following the strategy for knock-in Natsume et al. (2016) (link), to obtain the homology-directed repair (HDR) template, homology arms (HA) were amplified by PCR (sequences of oligonucleotides are listed in Table S4) using U2OS genomic DNA as template (5′HA, 400 bp; 3′HA, 800 bp). PCR fragments were digested with AgeI/EcoRI (5′HA) or BamHI/SpeI (3′HA) and cloned into the entry vector to obtain pJET-SPT6_HDR (5′HA-AID-V5-P2A-Blast-TGA-3′HA). To construct CRISPR/Cas9 vectors, two sgRNA were cloned into PX458 (Addgene #48138) as described (Ran et al., 2013 (link)) to obtain PX458_SPT6_sgR1 and PX458_SPT6_sgR2. 9x-myc-tagged TIR1 was PCR amplified using the template vector pBABE TIR1-9myc (Addgene #64945) and inserted into pRRL-hygro using AgeI/MluI digestion to obtain pRRL-hygro-TIR1.

Narain A., Bhandare P., Adhikari B., Backes S., Eilers M., Dölken L., Schlosser A., Erhard F., Baluapuri A, & Wolf E. (2021). Targeted protein degradation reveals a direct role of SPT6 in RNAPII elongation and termination. Molecular Cell, 81(15), 3110-3127.e14.

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Porcine IGF2 CRISPR Editing Protocol

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Guide sequence for sgRNA targeting the ZBED6 binding motif in intron 3 of porcine IGF2 were designed using the open-source tool, CHOPCHOP (http://chopchop.cbu.uib.no/). The BE3 (a fusion protein of deaminase Apobec-1, Cas9 (D10A) nickase, and Uracil Glycosylase Inhibitor (UGI)) coding sequence was digested from the pCMV-BE3 (Addgene #73021, Cambridge, MA, USA), and cloned into the pX458 (Addgene #48138, Cambridge, MA, USA) to replace the 3 × FLAG-Cas9 coding sequence to generate the pX458-BE3 plasmid, which contains the enhanced green fluorescent protein (EGFP) reporter. Oligos of gRNA targeting porcine IGF2-intron3-3071 were synthesized (Sangon Biotech, Shanghai, China) and cloned into the plasmid pX458-BE3 through Bbs I restriction site to create the plasmid pX458-BE3-gRNA.

Duo T., Liu X., Mo D., Bian Y., Cai S., Wang M., Li R., Zhu Q., Tong X., Liang Z., Jiang W., Chen S., Chen Y, & He Z. (2023). Single-base editing in IGF2 improves meat production and intramuscular fat deposition in Liang Guang Small Spotted pigs. Journal of Animal Science and Biotechnology, 14, 141.

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CRISPR-Cas9 Lck Knockout and Lck-TurboID Fusion

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Guide RNAs targeting an exon region of Lck were designed and cloned into pSpCas9(BB)-2A-GFP vector or PX458 (Addgene, 48138) for targeted deletion of Lck using CRISPR-Cas9 ^{20 (link)}. Lck and TurboID cDNA were amplified by PCR from pCMV-SPORT6-Lck (antibodies-online Inc, ABIN3804434) and V5-TurboID-NES_pCDNA3 (Addgene, 107169), respectively, using Phusion polymerase and 10 μM forward and reverse primers. PCR products were verified by gel electrophoresis and purified using Monarch PCR & DNA Cleanup Kit (New England BioLabs, T1030). Purified PCR products were digested accordingly and ligated into pEF6 mammalian expression vector (Thermo Fisher Scientific, V96120) to form pEF6-Lck-TurboID. Plasmid sequences were verified by DNA sequencing. All oligonucleotides used in this work were purchased from Sigma-Aldrich and are listed in Table S1. Plasmids PX458-Lck (#159430) and pEF6-Lck-TurboID (#159433) are deposited to Addgene.

Chua X.Y., Aballo T., Elnemer W., Tran M, & Salomon A. (2020). Quantitative interactomics of Lck-TurboID in living human T cells unveils T cell receptor stimulation-induced proximal Lck interactors. Journal of proteome research, 20(1), 715-726.

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CRISPR-Based PoMaf1 Knockout

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Using the CRISPR RGEN Tools (www.rgenome.net/cas-designer), a single guide RNA (sgRNA) targeting PoMaf1 was designed (5′-CAA CAC ACA GCT GGG AGC TGA GG-3′; PAM, underlined). A pair of oligonucleotides corresponding to the target sgRNA sequence (oligo-1, 5′-CAC CGC AAC ACA CAG CTG GGA GCT G-3′; oligo-2, 5′-AAA CCA GCT CCC AGC TGT GTG TTG-3′) was inserted into the sgRNA/Cas9 dual expression vector pSpCas9(BB)-2A-GFP (also known as PX458; Addgene, Watertown, MA, USA) according to the protocol of Zhang [50 (link)]. Briefly, the PX458 vector was digested with BbsI and gel-purified. The dsDNA with 4 bp overhangs on both ends was generated by annealing following phosphorylation, and ligated into the linearized PX458 vector.

Kim J., Cho J.Y., Kim J.W., Kim D.G., Nam B.H., Kim B.S., Kim W.J., Kim Y.O., Cheong J, & Kong H.J. (2021). Molecular Characterization of Paralichthys olivaceus MAF1 and Its Potential Role as an Anti-Viral Hemorrhagic Septicaemia Virus Factor in Hirame Natural Embryo Cells. International Journal of Molecular Sciences, 22(3), 1353.

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CRISPR Knockout of MIGA2 in HeLa Cells

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CRISPR gRNAs targeting the third exons of MIGA2 were designed using the online TKO CRISPR Design Tool (https://crispr.ccbr.utoronto.ca/crisprdb/public/library/TKOv3/). 5′-GCGGAAAGTCCTCTTTGCCA-3′ was chosen as gRNA to knock out MIGA2. The gRNA was cloned into pX458 (48138; Addgene) as described previously (Ran et al., 2013 (link)). Hela cells were transiently transfected with the constructs containing gRNAs. After 48 h, the GFP positive individual cells were selected by flow cytometry (BD FACSAria) and seeded in 96-well plates for single clones. The clones were validated by genotyping and Western blotting. For genotyping, briefly, genomic DNAs from single clones were extracted using QuickExtract DNA extraction solution (QE0905T; Lucigen), and PCR products containing the site of Cas9 cleavage site were generated using the following primers: 5′-TAGACCTCACCTTCTCGGCACT-3′; 5′-CCAATATCCCCAAGTAGAGAGTG-3′. PCR products were sequenced.

Hong Z., Adlakha J., Wan N., Guinn E., Giska F., Gupta K., Melia T.J, & Reinisch K.M. (2022). Mitoguardin-2–mediated lipid transfer preserves mitochondrial morphology and lipid droplet formation. The Journal of Cell Biology, 221(12), e202207022.

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Genetic Manipulation of Mouse Epigenetic Regulators

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sgRNAs directed against mouse Dnmt3a, Nsd1, Nsd2, and Setd2 were cloned into px458 (Addgene #48138, a gift from Feng Zhang). Mouse Dnmt3a and Dnmt3b cDNA sequences from Horizon Dharmacon were cloned into pCDH-EF1-MCS-Neo and piggybac (pCAGGS-IRES-Neo, a gift from H. Niwa, Institute of Molecular Embryology and Genetics, Kumamoto, Japan) with an N-terminal FLAG-HA epitope tag using Gibson assembly (NEB). Mouse Nsd1 cDNA from Horizon Dharmacon was cloned into pPB-CAG-3xFLAG-empty-pgk-hph (Addgene #48754, a gift from Austin Smith) using Gibson assembly. Standard site-directed mutagenesis techniques were used to generate C2023A in Nsd1 and the TRBS missense mutations W297del, I310N and Y365C corresponding to mouse Dnmt3a residues W293, I306 and Y361 respectively. To produce lentivirus, 293T cells were transfected with the lentiviral vector and helper plasmids (psPAX2, pVSVG). Supernatant containing lentivirus was collected and filtered 48 hours later for transduction.

Weinberg D.N., Papillon-Cavanagh S., Chen H., Yue Y., Chen X., Rajagopalan K.N., Horth C., McGuire J.T., Xu X., Nikbakht H., Lemiesz A.E., Marchione D.M., Marunde M.R., Meiners M., Cheek M., Keogh M.C., Bareke E., Djedid A., Harutyunyan A.S., Jabado N., Garcia B.A., Li H., Allis C.D., Majewski J, & Lu C. (2019). H3K36me2 recruits DNMT3A and shapes the intergenic DNA methylation landscape. Nature, 573(7773), 281-286.

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Generation and Validation of PARP1/PARP2 Knockout Models

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Guides targeted to Parp1 and Parp2 genes were designed using the CRISPR design tool (http://crispr.mit.edu/). The best target guides were selected for each gene. gRNA sequences are as follows: PARP1, 5′-CACCGCACTCGATAAAGCCTCTCCG-3′ and 5′-AAACCGGAGAGGCTTTATCGAGTGC-3′; Parp2, 5′-CACCGCTTCAAGAGCGATGGCGCCG-3′ and 5′-AAACCGGCGCCATCGCTCTTGAAGC-3′. These guides were cloned into mammalian expression vector PX458 (Addgene). Mouse E14TG2a ESCs were transfected with Parp1 and/or Parp2 KO expressing guides and GFP-positive cells were sorted 48 h after transfection. The sorted cells were clonally expanded, and KO clones for Parp1, Parp2, and Parp1/Parp2 were verified by DNA sequencing, Western blots, and quantitative PCR.

Azad G.K., Ito K., Sailaja B.S., Biran A., Nissim-Rafinia M., Yamada Y., Brown D.T., Takizawa T, & Meshorer E. (2018). PARP1-dependent eviction of the linker histone H1 mediates immediate early gene expression during neuronal activation. The Journal of Cell Biology, 217(2), 473-481.

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Genome Editing of Alzheimer's Disease Models

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Genome-edited lines were generated using published protocols. Briefly, guide RNAs (gRNAs) to APP were generated using the Zhang Lab Crispr design Web site at MIT (crispr.mit.edu) and cloned into vector px458 (Addgene) that co-expresses the Cas9 nuclease and GFP. hiPSCs were electroporated with the px458 plasmid containing the gRNAs to target APP. We generated the APP KO cells from our parental APP^Dp cell line (APP^Dp1), first described in Israel et al., 2012 (link). To generate APP KO cells, no repair template was added. For generating the APP Swedish mutation, we targeted wild-type hiPSCs derived from J. Craig Venter (Gore et al., 2011 (link)) and 120-bp single-stranded oligonucleotide was designed to have the 2-bp Swedish mutation (GA to TC, corresponding to exon 16 of APP). Electroporated hiPSCs were sorted for GFP and plated a clonal density on 10-cm MEF plates. After 1.5 to 2.0 weeks, sub-cloned colonies were picked and transferred to a 96-well plate. Colonies were spit in duplicate and one set was maintained for cell line generation and the other was analyzed for the mutation of interest by Sanger Sequencing. All gene-edited lines were digitally karyotyped as euploid by the Illumina Infinium HumanCoreExomeBeadChip, as has been previously described (Young et al., 2015 (link))

Young J.E., Fong L.K., Frankowski H., Petsko G.A., Small S.A, & Goldstein L.S. (2018). Stabilizing the Retromer Complex in a Human Stem Cell Model of Alzheimer’s Disease Reduces TAU Phosphorylation Independently of Amyloid Precursor Protein. Stem Cell Reports, 10(3), 1046-1058.

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