To recapitulate the gene mutations found in the patients, guide RNAs targeting the first exon of MANF gene for deletion were designed using Benchling (https://benchling.com ). gRNAs with the highest quality score and lowest off-targets score were selected and purchased, alongside the RNP components (HiFi Cas9 protein, crRNA and tracrRNA), from Integrated DNA Technologies (IDT) and utilized according to the manufacturer’s recommended protocol. Two million undifferentiated stem cells were electroporated with the RNP complex using Neon Transfection system (Thermo Fisher, 1100 V, 20ms, two pulses), and plated on Matrigel-coated plates in E8 medium containing 10 μM ROCK inhibitor overnight. Afterwards, cells were single-cell sorted, expanded, and screened for the desired deletion using PCR. Positive clones were validated by Sanger sequencing and characterized for pluripotency and chromosomal integrity.
Hifi cas9 protein
Manufactured by Integrated DNA Technologies
The HiFi Cas9 protein is a high-fidelity variant of the Cas9 enzyme, which is a key component in the CRISPR-Cas9 gene editing system. The HiFi Cas9 protein exhibits reduced off-target effects compared to the wild-type Cas9 enzyme, making it a useful tool for precision genome editing applications.
Automatically generated - may contain errors
Lab products found in correlation
Tracrrna, by Integrated DNA Technologies (3 mentions)
Crrna, by Integrated DNA Technologies (2 mentions)
Neon transfection system, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Azd7648, by Selleck Chemicals (1 mentions)
P3 buffer, by Lonza (1 mentions)
Phenol red, by Merck Group (1 mentions)
Hifi cas9 protein, by Thermo Fisher Scientific (1 mentions)
Nuclease free duplex buffer, by Integrated DNA Technologies (1 mentions)
4 protocols using hifi cas9 protein
1
Generation of MANF Gene Knockout Stem Cells
2
High-Efficiency CRISPR Editing in CD34+ Cells
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The HiFi Cas9 protein was purchased from Integrated DNA Technologies (IDT) or Aldevron. Before electroporation, RNPs were complexed at a Cas9/sgRNA molar ratio of 1:2.5 at 25 °C for 10-20 min. Next, CD34 + cells were resuspended in P3 buffer (Lonza) with complexed RNPs and subsequently electroporated using the Lonza 4D-Nucleofector and 4D-Nucleofector X Unit (program DZ-100). Electroporated cells were then plated at 1 × 10 5 -5 × 10 5 cells ml -1 in the previously described cytokine-supplemented media. Immediately after electroporation, AAV6 was dispensed onto cells at 2.5 × 10 3 -5 × 10 3 vector genomes per cell based on titre determined by ddPCR. For multiplex editing experiments, in addition to the steps described above, cells were incubated with 0.5 μM of the DNA-PKcs inhibitor AZD7648 (catalogue number S8843; Selleck Chemicals) for 24 h, as previously described 32, (link)33 .
3
MANF Gene Deletion in Stem Cells
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To recapitulate the gene mutations found in the patients, guide RNAs targeting the first exon of MANF gene for deletion were designed using Benchling (https://benchling.com ). Guide RNAs with the highest quality score and lowest off-target score were selected and purchased, alongside the RNP components (HiFi Cas9 protein, crRNA, and tracrRNA), from Integrated DNA Technologies and used according to the manufacturer’s recommended protocol. Two million undifferentiated stem cells were electroporated with the RNP complex using the Neon Transfection system (1,100 V, 20 ms, 2 pulses) (Thermo Fisher Scientific) and plated on Matrigel-coated plates in Essential 8 medium containing 10 μmol/L ROCK inhibitor overnight. Afterward, cells were single-cell sorted, expanded, and screened for the desired deletion using PCR. Positive clones were validated by Sanger sequencing and characterized for pluripotency and chromosomal integrity.
4
CRISPR-Cas9 RNP Assembly Protocol
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The chemically synthesized Alt-R-modified crRNA, tracrRNA, Hifi Cas9 protein, and nuclease-free duplex buffer were ordered from Integrated DNA Technologies. The crRNA and tracrRNA powders were diluted to 100 μM with nuclease-free duplex buffer. The 10 μl Hifi Cas9 protein was aliquoted into 10 tubes followed by 1:5 dilution with Opti-MEM (31985062; Thermo Fisher Scientific) solution before use.
Next, we prepared a 10 μM crRNA: tracrRNA duplex solution by mixing 1 μl crRNA stock solution, 1 μl tracrRNA stock solution, and 8 μl nuclease-free duplex buffer and then incubating at 95°C for 3 min in a thermocycler followed by natural cooling at room temperature for 15 min. Afterwards, we prepared the Cas9/gRNA RNP by mixing 2 μl Cas9 protein solution and 2 μl crRNA:tracrRNA duplex solution in 37°C for 10 min. Lastly, we mixed 2 μl Cas9/gRNA RNP, 5 μl donor dsDNA, and 0.8 μl phenol red (P0290; Sigma-Aldrich) and stored it at 4°C. We recommend performing this preassembly step the day before injection.
Next, we prepared a 10 μM crRNA: tracrRNA duplex solution by mixing 1 μl crRNA stock solution, 1 μl tracrRNA stock solution, and 8 μl nuclease-free duplex buffer and then incubating at 95°C for 3 min in a thermocycler followed by natural cooling at room temperature for 15 min. Afterwards, we prepared the Cas9/gRNA RNP by mixing 2 μl Cas9 protein solution and 2 μl crRNA:tracrRNA duplex solution in 37°C for 10 min. Lastly, we mixed 2 μl Cas9/gRNA RNP, 5 μl donor dsDNA, and 0.8 μl phenol red (P0290; Sigma-Aldrich) and stored it at 4°C. We recommend performing this preassembly step the day before injection.
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