Electroporation is an established method for transforming the green alga Chlamydomonas reinhardtii. Electroporators apply short (<~100 ms) electrical pulses that create transient pores in a cell's plasma membrane. Charged molecules (DNA, RNA, proteins) in close proximity to the plasma membrane can become embedded in the membrane after these transient pores have formed [2] (link). After several hours, the charged molecules may completely cross the plasma membrane and enter the cell. Genome edit rates using electroporation for CRISPR/Cas9 have been low [3 (link),4] (link). But recently, a high-efficiency protocol has been developed to electroporate Cas9 and sgRNA expression vectors into a wall-less strain (CC-3403) of C. reinhardtii. Under optimal conditions, 16% of transformants selected for antibiotic resistance contained Cas9-generated mutations as targeted by guide RNAs [1] (link). However, the apparatus used to perform the electroporation in this efficient protocol (NEPA21 Super Electroporator, NEPA Gene Co.) is quite expensive and not widely available. The goal of this work was to determine whether more commonly available electroporators could produce the high transformation efficiencies achieved by the NEPA21.
Nepa21 super electroporator
Manufactured by Nepa Gene
Sourced in Japan
The NEPA21 Super Electroporator is a laboratory instrument designed for the efficient transfer of genetic material, such as DNA or RNA, into cells. It utilizes electrical pulses to temporarily increase the permeability of cell membranes, allowing the introduction of the desired genetic material. The device features adjustable voltage and pulse duration settings to optimize the electroporation process for a variety of cell types.
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Lab products found in correlation
Opti mem, by Thermo Fisher Scientific (30 mentions)
Cas9 protein, by Thermo Fisher Scientific (4 mentions)
Cuy505p5, by Nepa Gene (3 mentions)
Cuy650p5, by Nepa Gene (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Cuy501p1 1, by Nepa Gene (2 mentions)
Opti mem media, by Thermo Fisher Scientific (2 mentions)
Mtesr1 medium, by STEMCELL (2 mentions)
Hyaluronidase solution, by Merck Group (2 mentions)
Crispr rna crrna, by Merck Group (2 mentions)
Tracrrna05n 5nmol, by Merck Group (2 mentions)
Facscanto 2 analyzer, by BD (2 mentions)
Arvo luminometer, by PerkinElmer (2 mentions)
Alt r xt crispr rna crrna, by Integrated DNA Technologies (2 mentions)
Nuclease free duplex buffer, by Integrated DNA Technologies (2 mentions)
Alt r cas9 nuclease v3 61μm, by Integrated DNA Technologies (2 mentions)
Fast green fcf, by Merck Group (2 mentions)
Nano glo dual luciferase reporter assay system, by Promega (2 mentions)
Tracrrna, by Integrated DNA Technologies (2 mentions)
Control sirna 1027280, by Santa Cruz Biotechnology (2 mentions)
91 protocols using nepa21 super electroporator
1
Efficient CRISPR Editing in Chlamydomonas
2
QSOX2 Knockdown Using siRNA Transfection
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Negative control siRNA (Bioneer, Daejeon, Republic of Korea) and QSOX2 siRNA were utilized for knockdown experiments. The cited sequence of #1 QSOX2 siRNA is as follows: Sense: GCAGCCAUUACGUGGCUAUtt, AntiSense: aaAUAGCCACGUAAUGGCUGC [5 ]. The pre-designed and combined #2 QSOX2 siRNA was purchased from Bioneer (169714-1, 169714-2, 169714-3, Bioneer, Daejeon, Republic of Korea). Each siRNA was transfected using Lipofectamine RNAiMAX Transfection Reagent (13778030, Invitrogen, MA, USA) according to the manufacturer's instructions. Alternatively, siRNA transfection was performed using the NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan) via cuvette electroporation, according to the manufacturer's instructions. The specific electroporation conditions for the poring pulse and transfer pulse are listed in Supplementary Table 2 .
3
CRISPR-Cas9 Zygote Electroporation
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NEPA21 Super Electroporator (NEPAGENE, Chiba, Japan) and 1 mm gap electrode (CUY501P1-1.5, NEPAGENE, Chiba, Japan) were used for electroporation. Zygotes were washed with Opti-MEM (Thermo Fisher Scientific) and then placed in the electrode gap filled with 5 µl of Opti-MEM solution containing 200 ng/µl Cas9 protein and 6 pmol/µl gRNA (crRNA/tracrRNA complex). The eggs were then cultured in KSOM medium at 37 °C and 5%CO2 in an incubator until the two-cell stage.
4
CRISPR Electroporation of Zygotes
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Fertilized eggs were suspended in Opti-MEM (Gibco, 31985-070) in the presence of RNP and placed on microscope slides with platinum-plated electrodes (NEPA GENE, CUY505P5). RNP was delivered into zygotes using a NEPA 21 Super Electroporator (NEPA GENE) according to the recommended protocol. Four days after incubation of the eggs in KSOM medium at 37 °C, cells at the morula stage were harvested for preparation of genomic DNA.
5
CRISPR/Cas9 Electroporation in Fertilized Eggs
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The gRNA was prepared as described above. The gRNA was mixed with Cas9 protein solution and opti‐MEM media (Thermo Fisher Scientific), and then incubated at 37 °C for 5 min to prepare the gRNA/Cas9 RNPs [final concentration: 50 ng/μL (≈ 330 nM) Cas9 for 20 ng/μL (≈ 600 nM) of each gRNA (Table S2 )]. The gRNA/Cas9 RNP solution was placed between the electrodes with a 5 mm gap in the NEPA21 Super Electroporator (Nepagene, Ichikawa, Chiba, Japan). Fertilized eggs were arranged between the electrodes, and then, the electroporation was done with the following conditions [resistance value: 550~600 Ω, poring pulse: 225 V (voltage), 2 ms (pulse amplitude), 50 ms (pulse interval), four (pulse number), 10% (attenuation), + (polarity), transfer pulse: 20 V (voltage), 50 ms (pulse amplitude), 50 ms (pulse interval), ± 5 (pulse number), 40% (attenuation), +/− (polarity)].
6
Electroporation of Cas9-gRNA RNPs in Fertilized Eggs
7
Genome Editing of Human Embryonic Stem Cells
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H1 hESCs were transfected with ZFN-L (5 μg), ZFN-R (5 μg), pAAV-Neo_Cas9 (2.5 μg), and pAAV-Puro_siKO (2.5 μg) plasmids with a NEPA21 Super Electroporator (Nepa Gene, Chiba, Japan; poring pulse: 150 V, transfer pulse: 20 V). The transfected cells were placed on a Matrigel-coated culture dish and incubated in mTeSR1 medium (STEMCELL Technologies Inc., Vancouver, Canada) containing 10 μM Y27632 (ROCK inhibitor, A. G. Scientific, San Diego, CA) at 37 °C and 5% CO2 for 3 d until they reached confluence. For transgenic colony selection, the cells were treated with 200 μg/ml G418 (InvivoGen, Pak Shek Kok, Hong Kong) for 4 d and then with 0.5 μg/ml puromycin (Sigma, St. Louis, MO) for 3 d. To expand the hESCs, each ESC colony was mechanically divided into 20–25 clumps, treated with 10 μg/ml dispase (Invitrogen, Carlsbad, CA) at 37 °C for 4 min, and divided into 3–4 culture dishes. All hESCs used in this study were mycoplasma-negative.
8
Generation of Catsperq knockout mice
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The Catsperq−/− mice were generated using CRISPR/Cas9-based gene targeting. Two guide RNAs gRNA1 (5′-accctggaattgtctcgggt-3′) and gRNA2 (5′-taagtcaagtgtcaacgacc-3′) targeting the coding region of Catsperq were designed using CRISPR direct software (http://crispr.dbcls.jp/ ) (45 (link)). Zygotes were isolated on the day of the coagulation plug (=E0.5) from superovulated female mice (B6D2F1) mated with B6D2F1 males. To remove cumulus cells, zygotes were incubated in hyaluronidase solution (0.33 mg/mL) (Sigma-Aldrich). Ribonucleoprotein complexes containing synthesized CRISPR RNA (crRNA) (Sigma-Aldrich), transactivating crispr RNA (tracrRNA) (TRACRRNA05N-5NMOL, Sigma-Aldrich), and CAS9 protein (A36497, Thermo Fisher Scientific) were electroporated into fertilized eggs using a NEPA21 superelectroporator (NEPA GENE) (46 (link)). Electroporated zygotes were incubated in potassium simplex optimization medium (47 (link)) at 37 °C and 5% CO2 until the next day. Two-cell embryos were transferred into the oviducts of pseudopregnant recipient females. The resulting pups were genotyped using primers TMEM249_F (5′-tgtggtcaatagaaaagcccct-3′), TMEM249_R (5′-cgcgtctcctcccacaagtac-3′) and TMEM249_R′ (5′-aaaggaggccagggctcaggcccca-3′).
9
Monitoring DNA Repair in HEK293A Cells
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HEK293A cells were treated with DSF/DMSO for 24 h. Cells were continued to electroporated with the I-SceI expression construct (pCBASce) together with DR-GFP or EJ5-GFP reporter plasmid at 150 V, 975 μF using NEPA21 Super Electroporator (NEPA GENE). Cells were further recovered for 48 h after electroporation and were subjected for flow cytometric analysis using a BD FACS CantoII Analyzer.
10
DNA Immunization and Electroporation in Mice
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C57BL/6 (5–7 animals/group) or AB6 (4–6 animals/group) mice were immunized with 2 doses of 50 μg of pUMVC3, pNS1-ZIKV, or pgDNS1-ZIKV formulated in apyrogenic saline (0.9%). For immunization, the animals were previously anesthetized with a mixture of Ketamine and Xylazine (100 and 10 mg/kg, respectively) administered intraperitoneally (i.p.). Then, the animals received intramuscular (i.m.) injections with the formulations followed by electroporation (two 130 V pulses with 1 ms duration and four 70 V pulses with 50 ms duration, with an interval of 450 ms between each pulse), which was carried out with the CUY560-5-0.5 electrode using the NEPA21 Super Electroporator (Nepa Gene Co, Japan.), at an interval of 2 weeks. Blood samples were obtained by submandibular plexus puncture 14 days after the administration of each dose and centrifuged at 3,000 g for 30 min to separate the sera. The obtained serum samples were stored at −20°C for future analysis.
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