Pre-designed DNA primers for Bub1 guide RNA template assembly were purchased from GeneArt (Thermo Fisher) with forward primer IVT-TAATACGACTCACTATAGTACAAGGGCAATGACC-T1 and reverse primer IVT-TTCTAGCTCTAAAACAGAGGGTCATTGCCCTTGT-T1. Guide RNA was synthesized according to the instruction using the GeneArt Precision gRNA Synthesis kit (Thermo Fisher). An amount of 625 ng of guide RNA and 2,500 ng of Cas9 nuclease (GeneArt Platinum Cas9 Nuclease, Thermo Fisher) were transfected into 4.5 × 105 HeLa cells by Lipofectamine CRISPRMAX transfection reagent. Twenty-four hours later, the cells were split into a six-well plate with coverslips inside. Ninety-six hours after transfection, nocodazole (200 ng ml−1)-treated cells were fixed and immunofluorescence was performed as described above.
Geneart platinum cas9 nuclease
Manufactured by Thermo Fisher Scientific
Sourced in China, United States
The GeneArt Platinum Cas9 Nuclease is a recombinant Cas9 endonuclease derived from Streptococcus pyogenes. It is designed for use in CRISPR-Cas9 genome editing applications.
Automatically generated - may contain errors
Lab products found in correlation
Geneart precision grna synthesis kit, by Thermo Fisher Scientific (7 mentions)
Opti mem 1, by Thermo Fisher Scientific (4 mentions)
Injectman ni2, by Eppendorf (3 mentions)
Femtojet, by Eppendorf (3 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Precision grna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Minute total protein extraction kit, by Invent Biotechnologies (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Neon transfection kit, by Thermo Fisher Scientific (1 mentions)
Neon transfection system, by Thermo Fisher Scientific (1 mentions)
Tracrrna, by Thermo Fisher Scientific (1 mentions)
Nepa21, by Nepa Gene (1 mentions)
Nanoject 3, by Drummond (1 mentions)
Geneart crispr grna design tool, by Thermo Fisher Scientific (1 mentions)
Neon system, by Thermo Fisher Scientific (1 mentions)
Mmessage mmachine t7 transcription kit, by Thermo Fisher Scientific (1 mentions)
Megaclear, by Thermo Fisher Scientific (1 mentions)
30 protocols using geneart platinum cas9 nuclease
1
Bub1 CRISPR Guide RNA Synthesis and Transfection
2
CRISPR-Cas9 Targeting of Sf-FGFR and Sf-SR-C
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The sgRNA of Sf-SR-C, Sf-FGFR gene was designed using sgRNA design software, and the off-target risk of sgRNA was evaluated using the whole genome of S. frugiperda as a reference sequence [34 (link)]. The N18NGG sequence with the highest score and no potential off-target sites was selected as the sgRNA target sequence. The Sf-FGFR target sequence (5′-TATGTGCAGCAGAAACATTGG -3′, the PAM sequence is underlined) at exon 1 of Sf-FGFR, and the Sf-SR-C target sequence (5′-TGTACTGTGATGGATCCAATTGG -3′, the PAM sequence is underlined) at exon 1 of Sf-SR-C. SgRNA was synthesized in vitro according to the instructions of the GeneArt™ Precision gRNA Synthesis Kit. Then the sgRNA was purified by the gRNA Clean Up Kit. Cas9 protein (GeneArt ™ Platinum ™ Cas9 Nuclease) was purchased from Thermo Fisher Scientific (Shanghai, China).
3
Generation of PHYHIPL Ser19Stop Knock-in Mice
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We generated the PHYHIPL Ser19Stop knock-in mice using the electroporation method according to our previous report with some modifications [20 (link)]. In this study, we used synthetic crRNA (Alt-R® CRISPR-Cas9 crRNA; IDT, Coralville, IA, USA) and tracrRNA (Alt-R® CRISPR-Cas9 tracrRNA, IDT) instead of single guide RNA (sgRNA). We also designed the corresponding donor single-stranded oligodeoxynucleotides (ssODNs) with 5′- and 3′-homology arms. Target sequence: CATATCCCATGAGATCTTGAAGG. Equal volumes of crRNA and tracrRNA were combined in a duplex buffer (IDT) in a thermal cycler at 95 °C for 5 min following the manufacturer’s protocol. crRNA/tracrRNA (3 μM), recombinant Cas9 protein (100 ng/μL; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA, USA) and ssODN (400 ng/μL; Ultramer®, IDT) were mixed in Opti-MEM I (Thermo Fisher Scientific) before electroporation. Fertilized eggs were isolated from the superovulated C57BL/6 J female mice 21 h after administering human chorionic gonadotropin (hCG). The electroporation was conducted at the 1-cell stage (24–26 h after hCG), and the 2-cell stage embryos were transferred to the oviduct of pseudopregnant ICR females (Charles River Japan, Yokohama, Japan). We confirmed the mutant alleles of the obtained mice (Fig. 1 d) by sequencing PCR products.
4
Endogenous Bub1 Knockout in HeLa Cells
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Recombinant Cas9 protein was purchased (GeneArt Platinum Cas9 Nuclease, Thermofisher). gRNA targeting Bub1 exon2 was synthesized according to the instructions with the pre-designed forward primer IVT-TAATACGACTCACTATAGTACAAGGGCAATGACC and reverse primer IVT-TTCTAGCTCTAAAACAGAGGGTCATTGCCCTTGT (GeneArt Precision gRNA Synthesis Kit, Thermofisher). Then, 625 ng of gRNA was incubated with 2500 ng of Cas9 nuclease to form the complex, which was transfected into HeLa cells with Lipofectamine CRISPRMAX reagent. The cells were re-cultivated for single-cell-clone isolation at 24 h after transfection. More than 20 clones were expanded and examined for the presence of endogenous Bub1 by western blotting or immunofluorescence.
5
Efficient Generation of DBR1 Knockout HEK293T Cells
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HEK293T cells were cultured in DMEM (Thermo Fisher Scientific) and 10% Fetal Bovine Serum (Gibco) on 10cm plates 24 hours prior to transfection at 40% confluency. The following were transfected to each plate: 3.6ug DBR1 sgRNA (5’-AGACGCTGGCGCTGGCAGAG-3’) or scramble control (Origene), 3.6ug GFP-puro donor DNA (Origene), 15ug GeneArt Platinum Cas9 nuclease (Thermo Fisher Scientific) and 43ul Lipofectamine 2000 (Thermo Fisher Scientific). At 48 hours post-transfection, cells were split 1:10, and again every 3 days (7 times in total). After which, cells were grown in 0.5ug/ml Puromycin for selection, and single colonies were expanded. Minute™Total Protein Extraction kit (Invent Biotechnologies) was used, and DBR1 knockout werewas confirmed on single colonies #19 (C19) and #22 (C22) by western blot with the following antibodies: rabbit polyclonal anti-DBR1 (Proteintech, Cat no : 16019–1-AP) at 1:500 and mouse monoclonal anti-GAPDH (Santa Cruz Biotechnology, sc-47724) at 1:200. Cells from single colonies C19 and C22 were further validated with genomic PCR, and RNA was extracted using TRIzol™ (Thermo Fisher Scientific) per manufacturer’s instruction and sent to Genewiz for library preparation and sequencing.
6
Microinjection of CRISPR Reagents into Eggs
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The collection and preparation of eggs were carried out as reported by Wang et al.23 (link) Briefly, fresh eggs laid within 2 h were washed down from the gauzes in 1% sodium hypochlorite solution and rinsed with distilled water. After suction filtration, the eggs were lined up on a microscope slide (fixed with double-sided adhesive tape).
About one nanoliter mix of sgRNA14 (100 ng/μl), sgRNA12 (100 ng/μl) and Cas9 protein (100 ng/μl, GeneArt™ Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Shanghai, China) were injected into individual eggs using a FemtoJet and InjectMan NI 2 microinjection system (Eppendorf, Hamburg, Germany). The microinjection was completed within 2 h. Injected eggs were placed at 26 ± 1 °C, 60 ± 10% RH for hatching.
About one nanoliter mix of sgRNA14 (100 ng/μl), sgRNA12 (100 ng/μl) and Cas9 protein (100 ng/μl, GeneArt™ Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Shanghai, China) were injected into individual eggs using a FemtoJet and InjectMan NI 2 microinjection system (Eppendorf, Hamburg, Germany). The microinjection was completed within 2 h. Injected eggs were placed at 26 ± 1 °C, 60 ± 10% RH for hatching.
7
CRISPR-Cas9 Editing of Corn Borer Eggs
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Mated female moths of O. furnacalis were allowed to lay egg masses on pieces of wax paper previously placed on the top of the mating cage. Fresh egg masses (within 2 h after oviposition) were immediately collected by cutting the wax paper. Then, the eggs were lined on double-sided adhesive tape on a microscope slide. About 1 nL of mixture containing 150 ng/μL of Cas9 protein (GeneArt™ Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Shanghai, China) and 300 ng/μL of sgRNA were injected into each egg using a FemtoJet and InjectMan NI 2 microinjection system (Eppendorf, Hamburg, Germany). After injection, the embryos were incubated in an incubator as described above. The hatched larvae were fed an artificial diet without any toxin.
8
CRISPR-Cas9 Genome Editing of Mecp2 and Tet3 Genes
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Two gRNAs targeting Mecp2 intron 2 and intron3 (Table S6 ) were designed, as well as corresponding lox site ssODNs with 60 bp homology to sequences on each side of each gRNA-mediated DSB (Table S6 ). To facilitate the detection of correct insertions, the ssODNs targeting intron 2 and the ssODN targeting intron 3 were engineered to contain a NheI restriction site and an EcoRI site, respectively, in addition to the lox sequences. Two gRNAs targeting Tet3 intron 7 and intron 9 (Table S6 ) and two loxP site ssODNs, containing a BamHI or a EcoRI restriction site with 60 bp homology sequences, were also designed (Table S6 ). gRNAs were synthesized as previously described16 (link). Recombinant Cas9 protein (100 ng/μl; GeneArt Platinum™ Cas9 Nuclease, Thermo Fisher Scientific, Waltham, MA), gRNA (24 ng/μl), and ssODNs (400 ng/μl) (Table S6 ) were mixed in RNase-free water for microinjection or in Opti-MEM I (Life Technologies, Carlsbad, CA) for electroporation.
9
Knockout of GST Cluster Using CRISPR/Cas9
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To further analyze the function of the GST cluster, we knocked out the GST cluster using the CRISPR/Cas9 system. A pair of sgRNAs were designed using the sgRNAcas9 design tool (Supplementary Table 1 )62 (link). sgRNA1 and sgRNA2 were located on GST_116 and GST_121, respectively. The sgRNA target sequences were checked in a search of the H. armigera genome using the sgRNAcas9 design tool, and no potential off-target sites were identified. A PCR-based approach was used to prepare sgRNA according to the manufacturer’s instructions (GeneArt Precision gRNA Synthesis Kit, Thermo Fisher Scientific, USA). The template DNA for the in vitro transcription of the sgRNAs was made using PCR-based fusion of two oligonucleotides with the T7 promoter (Target F: TAATACGACTCACTATAG + the target sequence; Target R: TTCTAGCTCTAAAAC + the reverse complementary sequence of the target). The PCR conditions were as reported by Jin et al.35 (link). The sgRNAs were synthesized by in vitro transcription using the GeneArt Precision gRNA Synthesis Kit, according to the manufacturer’s instructions. The Cas9 protein (GeneArt Platinum Cas9 Nuclease) was purchased from Thermo Fisher Scientific (Shanghai, China).
10
CRISPR-based CD26 Knockout in Primary T Cells
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Primary human T cells were activated for 3 days with aCD3/ICOS beads before CRISPR manipulation. On day 3, cells were debeaded and rested for 3 hours before electroporation. Two micrograms of CD26-specific single-guide RNA (sgRNA) (GenScript) was combined with 1 μg of GeneArt Platinum Cas9 Nuclease (Thermo Fisher Scientific) per 1 × 106 cells and incubated for 20 min at room temperature. During this time, cells were washed three times in PBS and then resuspended in T buffer (Neon Transfection Kit, Thermo Fisher Scientific) at 4 × 106 cells/100 μl. The sgRNA-Cas9 mixture was then added to the cells, followed by electroporation (2250 V, 20 ms, 1 pulse) using the Neon Transfection system (Thermo Fisher Scientific). Control cells were electroporated without the addition of sgRNA-Cas9. Cells were immediately returned to culture in prewarmed cell media without antibiotics but containing an additional 1% l -glutamine. Following an overnight rest, cells were resuspended in complete culture medium with IL-2 for normal culture.
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