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Px459 plasmid

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PX459 is a plasmid vector designed for CRISPR/Cas9-mediated genome editing. It contains the Cas9 gene and a sgRNA expression cassette, allowing for targeted gene knockouts or modifications.

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Lab products found in correlation

Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Mab3662, by Merck Group (1 mentions) Quikchange lightning multi site directed mutagenesis kit, by Agilent Technologies (1 mentions) Pet100 d topo vector, by Thermo Fisher Scientific (1 mentions) In fusion hd cloning, by Takara Bio (1 mentions) Esp3i, by Thermo Fisher Scientific (1 mentions) Miniprep kit, by Qiagen (1 mentions) One shot top10 chemically competent e coli, by Thermo Fisher Scientific (1 mentions) Megashortscript t7 transcription kit, by Thermo Fisher Scientific (1 mentions) Premix taq, by Takara Bio (1 mentions) Megaclear kit, by Thermo Fisher Scientific (1 mentions) Mmessage mmachine t7 ultra kit, by Thermo Fisher Scientific (1 mentions) Be4 gam, by Addgene (1 mentions) Pcmv abe7, by Addgene (1 mentions) Pcmv be3, by Addgene (1 mentions) Ampicillin sodium salt, by Thermo Fisher Scientific (1 mentions) Restriction endonuclease dpni, by Thermo Fisher Scientific (1 mentions) Milli q purification system, by Merck Group (1 mentions) Imidazole, by Merck Group (1 mentions) Turbofect cationic polymerbased transfection reagent, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

PX459 (104 citations) PSpCas9(BB)-2A-Puro (PX459) V2.0 plasmid (23 citations) PSpCas9(BB)-2A-Puro (PX459) plasmid (21 citations) PX459 V2.0 plasmid (6 citations) PSpCas9(BB)-2A-Puro (PX459) V2.0 (PX459) plasmid (2 citations)

16 protocols using px459 plasmid

1

CRISPR-Mediated Gene Knockout in HEK293A and B16-OVA Cells

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CRISPR genomic editing technology was used to delete genes in HEK293A and B16-OVA cells. The guide RNA sequences were cloned into the px459 plasmid (Addgene 48319). The constructed plasmids were transfected into HEK293A or B16-OVA using PolyJet. 24 h after transfection, the transfected cells were enriched by 2 μg/ml puromycin selection for 2–3 days and then were sorted into 96-well plates with only one cell in each well by FACS (UCSD; Human Embryonic Stem Cell Core, BDInflux). The clones were screened by western blot analysis with gene-specific antibodies. B16-OVA/LATS1/2 DKO, HEK293A/MST1/2 DKO and MAP4K4/6/7 TKO cells were generated as previously described38 (link),39 (link).
The single-guide RNA (sgRNA) sequences targeting individual genes were as follows:
human HSF-1#1: 5’-CAGCTTCCACGTGTTCGACC-3’;
human PP5#1: 5’-CGCGCTGCGAGACTACGAGA-3’;
human PP5#2: 5’-ACGCGCTGGGAGACGCCACG-3’;
human HSPA1A#2: 5’-AACCGGCATGGCCAAAGCCG-3’;
human HSPA1A#3: 5’-GGTGCTGGACAAGTGTCAAG-3’;
human HSPA1B#2: 5’-CACCGGCATGGCCAAAGCCG-3’;
human HSPA1B#3: 5’-GGTTCTGGACAAGTGTCAAG-3’;
human HSP90AA1#3: 5’-TTCTCTTGCAGGTGAACCTA-3’;
human HSP90AB1#3: 5’-CATTGCTATTTATTCCTCGT-3’;
mouse YAP1#2: 5’-GCCCAAGTCCCACTCGCGAC-3’,
mouse WWTR1#2: 5’-GCAGTGTCCCAGCCGAATCT-3’.
Luo M., Meng Z., Moroishi T., Lin K.C., Shen G., Mo F., Shao B., Wei X., Zhang P., Wei Y, & Guan K.L. (2020). Heat stress activates YAP/TAZ to induce the heat shock transcriptome. Nature cell biology, 22(12), 1447-1459.
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2

Generating Mov10 Knockout Cell Lines

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Guide RNAs (Additional file 14) were designed to the mouse Mov10 locus as described in [63 (link)] and cloned into pX459 plasmid (Addgene, Cambridge, MA, USA). Constructs were transfected into WT N2a, serially diluted into 96-well plates, and grown under puromycin (2 μg/mL) selection. Puromycin-resistant colonies were selected and screened for Mov10 expression using western blot analysis and confirmed by sequencing.
Skariah G., Seimetz J., Norsworthy M., Lannom M.C., Kenny P.J., Elrakhawy M., Forsthoefel C., Drnevich J., Kalsotra A, & Ceman S. (2017). Mov10 suppresses retroelements and regulates neuronal development and function in the developing brain. BMC Biology, 15, 54.
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3

CRISPR-Mediated Knockout of KAP1 and STING/MAVS

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Guide RNAs (sgRNAs, see Appendix Table S2) specific to several different exons of KAP1 (exons 1 and 9) were designed using the website: http://crispr.mit.edu/and cloned into the PX459 plasmid (Addgene), which was then transfected into HeLa and HEK293 T cells. After 24 h, the cells were subjected to puromycin selection for 24 h or until control cells had completely died. The bulk population was then used for single‐cell cloning by limiting dilution. Knockout was assessed across a panel of clones by Western blotting using the KAP1 antibody, MAB3662 (Millipore), and validated functionally using KAP1‐KZNF reporter assays. For knockout of STING or MAVS, sgRNAs (listed in Appendix Table S2) were cloned into lentiCRISPRv2 and THP‐1 cells transduced, selected with puromycin and single cell cloned as above.
Tie C.H., Fernandes L., Conde L., Robbez‐Masson L., Sumner R.P., Peacock T., Rodriguez‐Plata M.T., Mickute G., Gifford R., Towers G.J., Herrero J, & Rowe H.M. (2018). KAP1 regulates endogenous retroviruses in adult human cells and contributes to innate immune control. EMBO Reports, 19(10), e45000.
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4

Engineered ANKLE1 Constructs and Gene Knockouts

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Codons optimized ANKLE1615 (for expression in Escherichia coli) carrying an N‐terminal 6xHis tag cloned in pET100/D‐TOPO vector was purchased from GeneArt (Thermo Fisher Scientific). The ANKLE1669 cDNA cloned in pcDNA3.1+/N‐eGFP vector was purchased from Genescript (clone ID: OHU12449). The ANKLE1615, ANKLE1129‐615, ANKLE11‐420, ANKLE11‐360 and ANKLE11‐128 carrying a N‐terminal GFP tag were generated by PCR and cloned into pcDNA5/FRT/TO vector or pcDNA3.1+ vector. The catalytic‐dead mutant of ANKLE1, ANKLE1Y453A, was generated using a QuikChange Lightning Multi Site‐Directed Mutagenesis kit (Agilent). To generate the sgRNA vectors for gene knockout, pairs of annealed oligonucleotides were cloned into the pX459 plasmid (Addgene 62988) according to the published protocol.[44] The following sequences of sgRNA oligonucleotides were used for gene targeting:
ANKLE1: 5’‐CACCGTCCGCGTGTCGAGATCCTGC‐3’ and 5’‐AAACGCAGGATCTCGACACGCGGAC‐3’
GEN1: 5’‐CACCGCACATCCCCTTGCGTAATCT‐3’ and 5’‐AAACAGATTACGCAAGGGGATGTGC‐3’[45]MUS81: 5’‐CACCGTCTGAAATACGAAGCGCGTG‐3’ and 5’‐AAACCACGCGCTTCGTATTTCAGAC‐3’[45]TREX1: 5’‐CACCGGAGCCCCCCCACCTCTC‐3’ and 5’‐AAACGAGAGGTGGGGGGGCTCC‐3’[11]STING: 5’‐CACCGCATATTACATCGGATATCTG‐3’ and 5’‐AAACCAGATATCCGATGTAATATGC‐3’
Jiang H., Kong N., Liu Z., West S.C, & Chan Y.W. (2023). Human Endonuclease ANKLE1 Localizes at the Midbody and Processes Chromatin Bridges to Prevent DNA Damage and cGAS‐STING Activation. Advanced Science, 10(12), 2204388.
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5

CRISPR-Mediated Gene Knockout in HEK293A and B16-OVA Cells

Cited 2 times
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CRISPR genomic editing technology was used to delete genes in HEK293A and B16-OVA cells. The guide RNA sequences were cloned into the px459 plasmid (Addgene 48319). The constructed plasmids were transfected into HEK293A or B16-OVA using PolyJet. 24 h after transfection, the transfected cells were enriched by 2 μg/ml puromycin selection for 2–3 days and then were sorted into 96-well plates with only one cell in each well by FACS (UCSD; Human Embryonic Stem Cell Core, BDInflux). The clones were screened by western blot analysis with gene-specific antibodies. B16-OVA/LATS1/2 DKO, HEK293A/MST1/2 DKO and MAP4K4/6/7 TKO cells were generated as previously described38 (link),39 (link).
The single-guide RNA (sgRNA) sequences targeting individual genes were as follows:
human HSF-1#1: 5’-CAGCTTCCACGTGTTCGACC-3’;
human PP5#1: 5’-CGCGCTGCGAGACTACGAGA-3’;
human PP5#2: 5’-ACGCGCTGGGAGACGCCACG-3’;
human HSPA1A#2: 5’-AACCGGCATGGCCAAAGCCG-3’;
human HSPA1A#3: 5’-GGTGCTGGACAAGTGTCAAG-3’;
human HSPA1B#2: 5’-CACCGGCATGGCCAAAGCCG-3’;
human HSPA1B#3: 5’-GGTTCTGGACAAGTGTCAAG-3’;
human HSP90AA1#3: 5’-TTCTCTTGCAGGTGAACCTA-3’;
human HSP90AB1#3: 5’-CATTGCTATTTATTCCTCGT-3’;
mouse YAP1#2: 5’-GCCCAAGTCCCACTCGCGAC-3’,
mouse WWTR1#2: 5’-GCAGTGTCCCAGCCGAATCT-3’.
Luo M., Meng Z., Moroishi T., Lin K.C., Shen G., Mo F., Shao B., Wei X., Zhang P., Wei Y, & Guan K.L. (2020). Heat stress activates YAP/TAZ to induce the heat shock transcriptome. Nature cell biology, 22(12), 1447-1459.
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6

Construction of STARR Reporter Constructs

Cited 1 time
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The AR expression construct was modified from the pGFP-AR plasmid (20 (link)) by replacing a NheI-BspTI fragment containing EGFP and 510 bp of human AR and replacing it with a PCR-amplified fragment with primer-introduced ATG, NheI and BspTI sites. Individual STARR reporter constructs were generated by digesting the human STARR-seq vector (21 (link)) with SalI and AgeI and subsequent insertion of fragments of interest by In-Fusion HD cloning (TaKaRa). Fragments of interest: positive control region (near IP6K3 gene, hg19: chr6:33 698 504–33 698 853), AQP3 enhancer (hg19: chr9:33 437 258–33 437 811) and the AQP3 enhancer with AR binding site mutations (AQP3-Deleted and AQP3-AGA → TGT) were ordered as a gBlock (IDT) or GeneStrand (Eurofins) (see Supplementary Table S1 for the sequence of regions).
Oligos (Supplementary Table S2) encoding guide RNAs to delete the region downstream of the AQP3 gene were designed using the CRISPR Design tool (22 (link)), annealed and cloned into BbsI digested PX459 plasmid (Addgene #62988, (23 (link))) to generate plasmids PX459-AQP3_214 and PX459-AQP3_216.
Kulik M., Bothe M., Kibar G., Fuchs A., Schöne S., Prekovic S., Mayayo-Peralta I., Chung H.R., Zwart W., Helsen C., Claessens F, & Meijsing S.H. (2021). Androgen and glucocorticoid receptor direct distinct transcriptional programs by receptor-specific and shared DNA binding sites. Nucleic Acids Research, 49(7), 3856-3875.
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7

Cloning CRISPR sgRNA into PX459 for 3T3 transfection

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The candidate sgRNA sequences were annealed and cloned into a PX459 plasmid (Addgene plasmid, 48139), after restriction digestion with Esp3I (Thermo Fisher Scientific, FD0454). PX459 vectors with candidate sgRNAs were then seeded in 6-well plates and transfected into NIH-3T3 cells (at 60% confluence) using Lipofectamine™ 3000 (Invitrogen, L3000075) according to the manufacturer's protocol. The transfected cells were selected using Puromycine (2.5 µg/mL) prior to use for subsequent studies.
Song X., Cui Y., Wang Y., Zhang Y., He Q., Yu Z., Xu C., Ning H., Han Y., Cai Y., Cheng X., Wang J., Teng Y., Yang X, & Wang J. (2022). Genome Editing with AAV-BR1-CRISPR in Postnatal Mouse Brain Endothelial Cells. International Journal of Biological Sciences, 18(2), 652-660.
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8

CRISPR-Mediated Knockout of p190A Gene

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The guide oligos targeting the p190A gene were subcloned into the pX459 plasmid (Addgene, USA). After 24 h, cells were cultured in 1 µg/ml puromycin for 3 days. Surviving cells were seeded in a 96-well plate by limited dilution to isolate the monoclonal cell lines. KO cell clones were detected by WB and validated by Sanger sequencing. The sequences of sgRNA and primers for amplification of the sgRNA-targeted sequence of the p190A gene are listed in Supplementary Tables 3 and 4.
Wen X., Wan J., He Q., Wang M., Li S., Jiang M., Qian Z., Liu B., Lu W., Wang K., Gao K, & Wan X. (2020). p190A inactivating mutations cause aberrant RhoA activation and promote malignant transformation via the Hippo-YAP pathway in endometrial cancer. Signal Transduction and Targeted Therapy, 5, 81.
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9

FAM83A Gene Knockout Protocol

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FAM83A-sgRNA/Cas9 plasmid vector was constructed using pX459 plasmid, which was purchased from Addgene (#48139). Two candidate sequences to target Mus musculus FAM83A were designed on the Zhangfeng Lab guide-design website (https://zlab.bio/guide-design-resources).
FAM83A-sgRNA1/Cas9 F: CACCGGGCAAGATCCGGAAACGTC
FAM83A-sgRNA1/Cas9 R: AAACGACGTTTCCGGATCTTGCCC
FAM83A-sgRNA2/Cas9 F: CACCGGTAGACTTCCTGTCCTCAG
FAM83A-sgRNA2/Cas9 R: AAACCTGAGGACAGGAAGTCTACC
The primer sequences of genotyping for the FAM83A-KO.
FAM83A-sgRNA/Cas9-g F: CTACGTCTGGAAGAGCTCCG
FAM83A-sgRNA/Cas9-g R: TCAGCCAAAGTCCAGGTGTG
Huang K., Jia Z., Li H., Peng Y., Chen X., Luo N., Song T., Wang Y., Shi X., Kuang S, & Yang G. (2022). Proto-oncogene FAM83A contributes to casein kinase 1–mediated mitochondrial maintenance and white adipocyte differentiation. The Journal of Biological Chemistry, 298(10), 102339.
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10

CRISPR-Cas9 Mediated UCHL1 Knockout in HEK293E and SH-SY5Y Cells

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The CRISPR-Cas9 system was used for the generation of UCHL1 KO cells19 (link). To generate UCHL1 KO HEK293E cells, the guide RNA sequence (GTGGCGCTTCGTGGACGTGC) was inserted into the PX459 plasmid (62988, Addgene). The vector was transfected into HEK293E cells. 2 days after transfection, the transfected cells were selected with puromycin (5 μg/ml) for 3 days. Then, single colonies were transferred onto 96-well plates with one colony in each well. Immunoblot analysis with anti-UCHL1 antibody (13179S, Cell signaling) was performed to screen UCHL1 KO clones. UCHL1 KO SH-SY5Y cells were generated in the previous paper19 (link).
Lee D., Yoon E., Ham S.J., Lee K., Jang H., Woo D., Lee D.H., Kim S., Choi S, & Chung J. (2024). Diabetic sensory neuropathy and insulin resistance are induced by loss of UCHL1 in Drosophila. Nature Communications, 15, 468.
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