Genome editing of frozen-warmed embryos was performed using the TAKE method18 (link)–20 (link). A super-electroporator NEPA21 (NEPA GENE Co. Ltd., Chiba, Japan) was used to introduce nucleases. The nuclease solution comprised 100 μg/mL of Cas9 protein (Integrated DNA Technologies Inc. Coralville, IA, USA) and 500 μg/mL of dual RNA (mixture of crRNA and tracrRNA, Integrated DNA Technologies Inc.) in Opti-MEM (Thermo Fisher Scientific Inc., MA, USA). crRNA was designed to target the tyrosinase gene in C57BL/6 mice (5′-GGGTGGATGACCGTGAGTCC-3′)32 (link). The nuclease solution (5 μl) was placed between metal plates of electrodes with a gap of 1 mm on a glass slide (CUY501P1-1.5, NEPA GENE Co. Ltd.). Embryos were placed in a line between electrodes. The poring pulse was set to voltage: 40 V, pulse length: 2.0 ms, pulse interval: 50 ms, number of pulses: 4, decay rate: 10%, polarity: + . The transfer pulse was set to a voltage: 15 V, pulse length: 50 ms, pulse interval: 50 ms, number of pulses: 5, decay rate: 40%, polarity: + / − . The genome editing rates of the offspring were estimated using the eye color difference (white: successful genome edition; black: not successful).
Cuy501p1 1
Manufactured by Nepa Gene
Sourced in Japan
The CUY501P1-1.5 is a laboratory equipment product. It is a precision instrument designed for use in scientific research and analysis. The core function of this product is to perform specific tasks within a controlled laboratory environment. No further details about the intended use or capabilities of this product can be provided in an unbiased and factual manner.
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Lab products found in correlation
6 protocols using cuy501p1 1
1
Genome Editing of Frozen Embryos
2
Zygote Electroporation for CRISPR-Cas12a Delivery
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Electroporations were carried out with a NEPA21 type II electroporator (Nepagene), a 5 µl electrode chamber with a gap width of 1 mm (Nepagene CUY501P1-1.5), and connection cables (Nepagene C115CB, and C117). The instrument was operated following the instructions of the manufacturer and used with the settings listed in Table 1 . The electrode chamber was filled with 5 µl of Cas12a nuclease pair mix. Zygotes (up to 40) were washed through 3 micro-drops of RNP mix and transferred into the electrode chamber by mouth pipetting. The sample impedance was measured on the NEPA21 instrument and adjusted to a value within the range from 190 and 200 Ohm by adding or removing RNP mix. After electroporation zygotes were transferred into center well culture dishes with KSOM medium supplemented with 1% NEAA.
Settings of the NEPA21 electroporation system for zygote electroporation using a 5 µl electrode chamber
| Voltage (V) | Length (ms) | Interval (ms) | Number of pulses | D. Rate (%) | Polarity | |
|---|---|---|---|---|---|---|
| Poring pulse | 30 | 3 | 100 | 6 | 10 | + |
| Transfer pulse | 5 | 50 | 50 | 5 | 40 | + / − |
3
Electroporation of Embryos for CRISPR
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The TAKE method was carried out to intact pronuclear stage embryos which collected at 22–24 h after hCG injection according to a previously described protocol
[7 (link)]. Super electroporator NEPA21 (NEPA GENE, Chiba, Japan) was used to introduce nucleases into embryos. The poring
pulse was set to voltage: 40 V, pulse length: 3.5, 2.0 or 0.5 msec, pulse interval: 50 msec, number of pulses: 4, decay rate: 10%, polarity: +. The transfer
pulse was set to a voltage: 15 V, pulse length: 50 msec, pulse interval: 50 msec, number of pulse: 5, decay rate: 40%, Polarity: +/–. The nuclease solution (5
μl) was filled between metal plates of 1 mm gap electrodes on a glass slide (CUY501P1-1.5; NEPA GENE). The embryos placed in line between the electrodes were
then discharged. The nuclease solution was exchanged for two operations to avoid dilution of the solution. After electroporation, the embryos were transferred
into KSOM.
[7 (link)]. Super electroporator NEPA21 (NEPA GENE, Chiba, Japan) was used to introduce nucleases into embryos. The poring
pulse was set to voltage: 40 V, pulse length: 3.5, 2.0 or 0.5 msec, pulse interval: 50 msec, number of pulses: 4, decay rate: 10%, polarity: +. The transfer
pulse was set to a voltage: 15 V, pulse length: 50 msec, pulse interval: 50 msec, number of pulse: 5, decay rate: 40%, Polarity: +/–. The nuclease solution (5
μl) was filled between metal plates of 1 mm gap electrodes on a glass slide (CUY501P1-1.5; NEPA GENE). The embryos placed in line between the electrodes were
then discharged. The nuclease solution was exchanged for two operations to avoid dilution of the solution. After electroporation, the embryos were transferred
into KSOM.
4
Efficient CRISPR/Cas9 Genome Editing in Mice
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Cas9 protein, crRNAs and tracrRNAs were purchased from Integrated DNA Technologies (Coralville, IA, USA). Two crRNAs were designed to target the Hr gene in eight inbred strains (5′-CTAACACTTGGCATGACCAA-3′ and 5′-GATGGAAGCCCCTGGCTAGA-3′). The RNP complex was prepared in Opti-MEM (Thermo Fisher Scientific, Waltham, MA, USA) with 2.4 μM Cas9 protein, 3.7 μM crRNA and 7.4 μM tracrRNA at 25°C for 5 min. RNP solutions were prepared immediately before electroporation and kept on ice until use.
Electroporation was performed using the TAKE method (Kaneko, 2017 (link)) with a NEPA21 Super Electroporator (NEPA GENE Co. Ltd, Chiba, Japan). The poring pulse was set to a voltage of 40 V, pulse length of 3.0 ms, pulse interval of 50 ms, number of four pulses, decay rate of 10% and + polarity. The transfer pulse was set to a voltage of 10 V, pulse length of 50 ms, pulse interval of 50 ms, number of five pulses, decay rate of 40% and +/− polarity. A 1-mm gap electrode (CUY501P1-1.5, NEPA GENE) was filled with 5 μl RNP solution, and zygotes washed with Opti-MEM solution were arranged on the electrode. Electroporated zygotes were observed for survival and cultured in KSOM overnight at 37°C and 5% CO2.
Electroporation was performed using the TAKE method (Kaneko, 2017 (link)) with a NEPA21 Super Electroporator (NEPA GENE Co. Ltd, Chiba, Japan). The poring pulse was set to a voltage of 40 V, pulse length of 3.0 ms, pulse interval of 50 ms, number of four pulses, decay rate of 10% and + polarity. The transfer pulse was set to a voltage of 10 V, pulse length of 50 ms, pulse interval of 50 ms, number of five pulses, decay rate of 40% and +/− polarity. A 1-mm gap electrode (CUY501P1-1.5, NEPA GENE) was filled with 5 μl RNP solution, and zygotes washed with Opti-MEM solution were arranged on the electrode. Electroporated zygotes were observed for survival and cultured in KSOM overnight at 37°C and 5% CO2.
5
CRISPR Delivery into Embryos
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Nucleases were introduced into pronuclear stage embryos 22–24 h after hCG injection using the TAKE method [7 (link)]. A super electroporator NEPA21 (NEPA GENE Co., Ltd., Chiba, Japan) was used to introduce the nucleases. The nuclease solution (5 μl) was placed between metal plates of 1 mm gap electrodes on a glass slide (CUY501P1-1.5; NEPA GENE Co., Ltd.). Embryos were placed in a line between the electrodes. The poring pulse was set to voltage: 40 V, pulse length: 0.5 or 3.5 msec, pulse interval: 50 msec, number of pulses: 4, decay rate: 10%, and polarity: +. The transfer pulse was set to voltage: 15 V, pulse length: 50 msec, pulse interval: 50 msec, number of pulses: 5, decay rate: 40%, and polarity: +/−. Embryos were then discharged and transferred into the HTF medium. The nuclease solution was exchanged for two operations to avoid dilution. Embryos placed in the nuclease solution without electroporation were used as controls.
6
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.
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