gRNA vectors were constructed using Uracil-Specific Excision Reagent (USER) friendly cloning as previously described (Ronda et al., 2014 (link)). The CHO codon-optimized dCas9 cassette was generated from a synthesized CHO codon-optimized wild-type Cas9 (GeneArt, Thermo Fisher Scientific) as previously described (Xiong et al., 2019 (link)). The CHO codon-optimized dCas9 was then fused to a VPR domain cloned from a AAV_NLS-SaCas9-NLS-VPR vector (a gift from George Church (Addgene plasmid #68496)). The construct is here referred to as VPR_dCas9 and available at Addgene (Addgene Plasmid #134601). The plasmid construct and sequence are listed in Supplementary Figure S3 . All plasmids were purified using NucleoBond Xtra Midi EF (Macherey-Nagel) according to manufacturer’s protocol and verified by Sanger sequencing.
Protocol detail
Construction of VPR_dCas9 Expression Plasmid
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Corresponding Organization : Novo Nordisk (United States)
Other organizations : Ajou University, Korea Advanced Institute of Science and Technology
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- Construction of gRNA vectors using Uracil-Specific Excision Reagent (USER) friendly cloning
- Generation of CHO codon-optimized dCas9 cassette from synthesized CHO codon-optimized wild-type Cas9
- Fusion of CHO codon-optimized dCas9 to a VPR domain cloned from an AAV_NLS-SaCas9-NLS-VPR vector
- Not explicitly mentioned
- Not explicitly mentioned
- Positive control: None mentioned
- Negative control: None mentioned
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