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Fig. 1 | Cell & Bioscience

Fig. 1

From: Multiplexed CRISPR/Cas9 gene knockout with simple crRNA:tracrRNA co-transfection

Fig. 1

CRISPR/Cas9 mediated gene KO via RNA oligo transfection. a Schematics for RNA duplex design. A target-specific crRNA 42-mer and a truncated tracrRNA 72-mer were used for the transfection reaction. The target specific 20-mer sequence in the crRNA is represented by degenerate Ns. b Transfection of an EGFP-targeted crRNA:tracrRNA duplex in a clonal 293T-EGFP1-Cas9 cell line that expresses both Cas9 and a single-copy EGFP transgene. Cells in 96 well plates were reverse transfected with increasing amount of RNA and lipid as indicated. The EGFP status of cells was analyzed by flow cytometry at day 5 and day 10 post-transfection. The “EGFP full” fraction indicates cells with EGFP signal similar to untreated cells; the “EGFP null” fraction indicates cells with no EGFP signal, and the “EGFP partial” fraction indicates cells with EGFP signal that are in between the full and null gates. c Transfection of an EGFP-targeted crRNA:tracrRNA duplex in a clonal U2OS-EGFP12-Cas9 cell line that expresses both Cas9 and 12 copies of the EGFP transgene. Cells in 24 well plates were reverse transfected with 25 nM of RNA. The EGFP status of cells was analyzed by flow cytometry at indicated time points (NT, non-targeting negative control crRNA)

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