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

Figure 1.

Figure 1

Cas9 protein delivery is allowed by guide RNA-encoding vectors

(A) Hypothetical genome editing model for Cas9 protein delivery with an all-in-one non-integrating lentiviral vector (NILV) encoding both enhanced green fluorescent protein (GFP)-targeting guide RNA and enhanced yellow fluorescent protein (YFP) donor DNA. The Cas9 protein was used with or without cyclophilin A (CypA) fusion for lentiviral packaging (CypA-Cas9 or Cas9-CypA). (B) Lentiviral titers at escalating doses of Cas9 fusion protein plasmids, evaluated by YFP expression in HeLa cells. Several silent mutations were added to the YFP gene to escape from GFP guide RNA targeting. (C) A GFP-to-YFP gene-conversion model using a GFP-positive human erythroleukemia (HEL) cell line (including a single copy of GFP gene) transduced with Cas9 protein delivery guide RNA/donor DNA vectors (all-in-one). GFP DNA breakage (GFP-YFP-), GFP-to-YFP gene conversion (GFP-YFP+), and false-positive conversion (GFP+YFP+) were evaluated by GFP- and YFP-positive percentages (% GFP and YFP). (D) GFP-to-YFP gene conversion using lentiviral Cas9 protein delivery with integrating guide RNA vectors or all-in-one NILVs at multiplicity of infection (MOI) 5, evaluated by flow cytometry 9 days post-transduction. (E) GFP-to-YFP gene conversion using all-in-one NILVs with CypA-Cas9 and Cas9-CypA fusion proteins as well as Cas9 protein alone (without CypA fusion), evaluated by flow cytometry 10 days post-transduction. (F) GFP DNA breakage 7 days post-transduction, evaluated by Surveyor nuclease assay using integrating or non-integrating guide RNA vectors (not encoding donor DNA) with protein delivery among CypA-Cas9, Cas9-CypA, and Cas9 without CypA fusion (intact: 0.8 kb, DNA break: 0.3 kb and 0.5 kb). (G) The all-in-one vector was separated into a GFP-targeting guide RNA vector and YFP donor DNA vector. Cas9 protein was delivered with either guide RNA vector or donor DNA vector. (H) GFP-to-YFP gene conversion using (1) guide RNA vector and Cas9 protein delivery donor DNA vector, and (2) Cas9 protein delivery guide RNA vector and donor DNA vector, evaluated by flow cytometry 9 days post-transduction. LTR, long terminal repeat; U6p, U6 promoter; Mp, murine stem cell virus promoter; gRNA, guide RNA. Values: mean ± standard error. All experiments were performed in triplicate except (F) (single run).