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. 2021 Mar 3;21:121–132. doi: 10.1016/j.omtm.2021.02.022

Figure 2.

Figure 2

More efficient genome editing with Cas9 protein delivery lentiviral vectors including multiple guide RNA sequences

(A) Design of lentiviral vectors encoding GFP-targeting guide RNA derived from a U6 promoter, multiple tandem copies (2×, 4×, 6×, and 9×) of pseudo guide RNA (the same GFP-targeting guide RNA with no additional promoter), and YFP donor DNA with Cas9 protein delivery. (B) Lentiviral vector titers for guide RNA/donor DNA (all-in-one) NILVs with Cas9 protein delivery including multiple pseudo guide RNA. (C) GFP-to-YFP gene conversion using these multiple pseudo guide RNA vectors at MOI 5, evaluated by flow cytometry 20 days post-transduction in a GFP+ HEL cell line. (D) GFP-to-YFP gene conversion at escalating MOIs (5, 10, 25, and 50) of guide RNA/donor DNA (all-in-one) NILV with Cas9 protein delivery without multiple pseudo guide RNA 14 days post-transduction. Values: mean ± standard error. All experiments were performed in triplicate.