Fig. 4: Packaging the DNA repair CRISPRa/i system with lentivirus for enhancement of HDR efficiency with viral delivery. | Cell Discovery

Fig. 4: Packaging the DNA repair CRISPRa/i system with lentivirus for enhancement of HDR efficiency with viral delivery.

From: Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9

Fig. 4

a The CDK1 activation plasmid was reconstructed into lentivirus backbone. Hygro, hygromycin. b HEK239FT cell was transduced with Cas9-Blast lentivirus to establish Cas9 constitutively expressed cell-line. Then the HEK239FT-Cas9 cell-line was transduced with dgCDK1-MS2:MPH lentivirus, followed by 2-3 days hygromycin selection. Finally, cells were transfected with sgAAVS1-Puro plasmid and SA-T2A-EGFP HR donor. The flow cytometry analysis was performed after 2 days' puromycin selection. Blast, blasticidin; Puro, puromycin. c The flow cytometry results demonstrated that HDR efficiency was significantly increased as compared with the vector group. d Schematic diagram representing the central idea of this study: with a single Cas9, through combinatorial usage of sgRNA and dgRNA for gene editing and CRISPRa/i on HDR/NHEJ machinery, HDR efficiency enhancement was achieved.

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