Pspcas9 bb 2a mirfp670

The DSB-inducer plasmid was constructed by ligation of two sgRNAs, targeting the EJ5-GFP cassette, into pX333 (Addgene #64073), and subsequent DrdI/KpnI-subcloning of the entire dual sgRNA expression cassette into pSpCas9(BB)-2A-miRFP670 (Addgene #91854). To run the EJ5-GFP assay, cells were transfected with 1 μg of this plasmid (Lipofectamine LTX, Invitrogen; 12-well format). After 30h cells were trypsinized, resuspended in 250 μL DPBS (Gibco), and analyzed on a
Canto II flow cytometer (BD Biosciences). Untransfected cells served as negative control to define proper gates in the APC and FITC channels for miRFP and GFP expression, respectively. DSB-repair negative cells were identified through Boolean gating, as shown in Fig. 3b. Flow cytometry data was analyzed in FlowJo (BD Biosciences) and Prism (GraphPad).

The DSB-inducer plasmid was constructed by ligation of two sgRNAs, targeting the EJ5-GFP cassette, into pX333 (Addgene #64073), and subsequent DrdI/KpnI-subcloning of the entire dual sgRNA expression cassette into pSpCas9(BB)-2A-miRFP670 (Addgene #91854). To run the EJ5-GFP assay, cells were transfected with 1 μg of this plasmid (Lipofectamine LTX, Invitrogen; 12-well format). After 30h cells were trypsinized, resuspended in 250 μL DPBS (Gibco), and analyzed on a Canto II flow cytometer (BD Biosciences). Untransfected cells served as negative control to define proper gates in the APC and FITC channels for miRFP and GFP expression, respectively. DSB-repair negative cells were identified through Boolean gating, as shown in Fig. 3b. Flow cytometry data was analyzed in FlowJo (BD Biosciences) and Prism (GraphPad).