CRISPR/Cas9 ribonucleoprotein (RNP) complex was formed by combining 1.5 µg of Cas9 protein (CAS9GFPPRO; Sigma) with 0.5 µg of chemically modified guide RNA (2′-O-methyl analogs and 3′ phosporothioate modifications were included at the three terminal nucleotides of the 5′ and 3′ ends; Sigma) for 10 min at room temperature. For HBG1/2 gene promoter editing, we used 1.5 µg of Cas9 with 0.5 µg of chemically modified sgRNA. For dual sgRNA-mediated HPFH3 genome editing, we used 1.5 µg of Cas9 with 0.25 µg of chemically modified sgRNA for target site-I sgRNA and 0.25 µg of chemically modified sgRNA for target site-II sgRNA. 0.5 × 106 BEL-A WT, BEL-A SCM and BEL-A BTM cells were electroporated using a Neon electroporation device as described above. The electroporated cells were immediately neutralized with a pre-warmed culture medium with cytokines and allowed to recover for 48 h before proceeding to erythroid differentiation.
Cas9gfppro
Manufactured by Merck Group
CAS9GFPPRO is a laboratory equipment product developed by Merck Group. It is a tool used for gene editing applications. The core function of CAS9GFPPRO is to facilitate the targeted modification of DNA sequences within cells.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using cas9gfppro
1
CRISPR/Cas9 Ribonucleoprotein Complex for Gene Editing
2
Cas9-GFP Protein CRISPR RNP Assay
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Cas9 from Streptococcus pyogenes, fused with enhanced GFP, recombinant, expressed in E. coli, 3X NLS was purchased from Sigma-Aldrich (CAS9GFPPRO). The sgRNAs used in this study were designed using E-CRISPR and MIT CRISPR design tool. Chemically modified sgRNAs (with 2′-O-Methyl at 3 first and last bases, and 3′ phosphorothiate bonds between first 3 and last 2 bases) were purchased from Synthego. For CRISPR/Cas9 RNP complexes assembly, CAS9GFPPRO was complexed to sgRNAs and incubated at room temperature for 15 min. For 1X concentration, 1250 ng CAS9GFPPRO were incubated with 7.5 pg sgRNAs in 5 μl buffer R (NeonTM, Thermo Fisher Scientific). For 2X condition, double concentration was used (Figure S1 A). The sgRNA sequences used in this study can be found in Table S2 .
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