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. 2020 Sep 10;14:570917. doi: 10.3389/fncel.2020.570917

FIGURE 1.

FIGURE 1

In vitro YFP sgRNA validation and selection. (A) YFP-targeting sequence for sgRNA design. YFP-targeting sgRNAs were designed (3 sgRNAs for SpCas9, 1 sgRNA for SaCas9, 2 sgRNAs for Cas12a, and 2 for CjCas9). (B) T7E1 assay to detect cleavage efficiency for YFP. Expected cleavage products by T7E1 were detected in 2% TAE gel. * Cleavage products around 590 and 260 bp. (C) Representative fluorescence microscopy images showing YFP expression in cells transfected with different CRISPR/Cas constructs. Scale bar: 100 μm. (D) Flow cytometry analysis for sgRNA selection. Data are represented as mean ± SEM for 4–7 independent replicates. Intergroup comparisons were performed using a one-way ANOVA and corrected for multiple comparisons. HEK293A cells without YFP expression were also included as negative control. No significant difference in YFP editing was observed between single and dual CRISPR/SaCas9 vector systems (p = 0.9608). Selected sgRNAs for in vivo testing were SpCas9 YFPsgRNA2, Cas12a YFP sgRNA20nt, and CjCas9 YFPsgRNA2. *p < 0.05, **p < 0.01.