Electroporation was performed using an Amaxa 4D-Nucleofector (Lonza, Morristown, NJ) using program CB-150. To improve survival, 10 μM Rho-associated protein kinase (ROCK) inhibitor Y27632 (StemRD Inc., Burlingame, CA) was added to the transfected cells. For FRT knock-in experiments, sgRNA expressing pX459 V2.0 plasmids and donor plasmid were used for nucleofection. Two days after electroporation, puromycin (0.5 μg/mL, Gibco, Gaithersburg, MD) was added to the medium for selection for 7–10 days. For Flpe-ERT2 knock-in and puromycin-resistant cassette removal, Cas9-2A-Cre, AAVS1-neo-CAG-FlpeERT2 (Addgene plasmid #68468 and #68460) [17 (link)], and sgRNA T2 (Addgene plasmid #41818) [20 (link)] were used for nucleofection. Two days after nucleofection, cells were treated with G418 (50 μg/mL, Corning) for 7–10 days and further treated with 100 μg/mL G418 for additional 10 days.
Sgrna t2
Manufactured by Addgene
The SgRNA T2 is a laboratory product designed for gene editing. It is a synthetic guide RNA that can be used in CRISPR-Cas9 systems to target and modify specific DNA sequences.
Automatically generated - may contain errors
2 protocols using sgrna t2
1
CRISPR-Based Gene Editing via Electroporation
2
Generation of AAVS1-pur-CAG-hM4Di-mCherry Donor Plasmid
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Human codon-optimized Streptococcus pyogenes wild-type Cas9 (Cas9-2A-GFP) and sgRNA T2 were obtained from Addgene (plasmid #44719, plasmid#41818)53 (link),54 (link). To generate AAVS1-pur-CAG-EGFP donor plasmid, we replaced the hrGFP gene in the AAVS1-pur-CAG-hrGFP plasmid (Addgene plasmid #52344)55 (link) with EGFP gene and inserted woodchuck hepatitis post-transcriptional regulatory element (WPRE) and human growth hormone (hGH) Poly A into the 3’ terminal of EGFP gene to obtain AAVS1-pur-CAG-EGFP donor plasmid. We next amplified hM4Di-mCherry cDNA by PCR from AAV-DIO-hM4Di-mCherry plasmid (a gift from Dr. Bryan L. Roth), respectively. hM4Di-mCherry was inserted into the AAVS1-pur-CAG-EGFP donor plasmid to replace EGFP to get the AAVS1-pur-CAG-hM4Di-mCherry donor plasmid.
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