High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System

Figures

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  Figure 1. Overview of the screening strategy. (a) Scheme of experimental procedures. (b) Effects of CRISPR working times on the off-target effects. (c) Scheme of in vitro screening to identify CRISPR/Cas9 modulators. (d) Vectors of CRISPR-based SSA.

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  Figure 2. High-throughput screening to identify compound modulators of CRISPR/Cas9 efficiency based on the single-strand annealing (SSA) assay. (a) Scheme of CRISPR-based SSA reporter system combined with dual luciferase (firefly luciferase and Renilla luciferase). (b) Relative luminescence of CRISPR-based SSA. HEK 293FT cells were divided into two groups, the experimental group was transfected with the CRISPR-based SSA reporter vector and pX330-B3, the control group was transfected with the SSA reporter vector alone. Forty-eight hours after transfection, the dual luciferase (firefly luciferase and Renilla luciferase) activities were detected by the Promega GloMax-Multi Instrument. The statistical significant differences were determined by Student’s t-test (*** p < 0.001). (c) The first-round screening to identify CRISPR/Cas9 modulators from 9930 compounds. (d) The second-round screening of the 640 compounds.

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  Figure 3. (a,b) The modulatory effects of CP-724714 and Clofarabine on the CRISPR/Cas9 efficiency, validated by relative luminescence reporter assays. The statistical significant differences were determined by Student’s t-test (** p < 0.01, *** p < 0.001). (c,d) Chemical structures of CP-724714 and Clofarabine. (e) Microscopic evaluation showing that the cytotoxic effects of the compounds were very low in HEK 293FT cells.

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  Figure 4. TALEN-based SSA reporter system. (a) Vectors of TALEN-based SSA. (b) Scheme of CRISPR-based SSA reporter system combined with dual luciferase (firefly luciferase and Renilla luciferase). (c) Relative luminescence of TALEN-based SSA. HEK 293FT cells were divided into two groups: the experimental group was transfected with TALEN-based SSA reporter vector and AAVS1- TALEN vector; the control group was transfected with TALEN-based SSA reporter vector alone. Forty-eight hours after transfection, the dual luciferase (firefly luciferase and Renilla luciferase) activities were detected by the Promega (GloMax-Multi Instrument). The statistical significant differences were determined by Student’s t-test (*** p < 0.001).

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  Figure 5. High-throughput screening to identify SSA decelerators and accelerators. (a) Line chart showing the SSA decelerators and accelerators identified by CRISPR or TALEN-based SSA reporter system. (b–e) The modulatory effects of Tranilast (b), Cerulenin (c), Rosolic acid (d) and Resveratrol (e) on the SSA efficiency validated by relative luminescence reporter assays (statistical significance was assessed using Student’s t-test. * p < 0.05; *** p < 0.001). (f) Chemical structures of Tranilast, Cerulenin, Rosolic acid and Resveratrol.