Skip to main content
. 2022 Feb 7;14:313–320. doi: 10.1016/j.bioactmat.2022.01.046

Fig. 5.

Fig. 5

Inducible Piggybac Cas13d system enables robust RNA editing.

A. Diagram of XLOne-Puro-Cas13d-eGFP-U6-gRNA plasmid design. B. Diagram of gRNA design to target THY1 gene and primer design for qPCR experiments. C. Dox addition induced GFP expression in the nucleus of IMR90C4 XLOne-Puro-Cas13d-eGFP-U6-THY1gRNA cells. Scale bar: 100 μm. D. Relative expression of THY1 RNA induced by different gRNA sequences. E. Flow cytometry of CD90 knockdown with different gRNA designs. F. Diagram of gRNA design to target SOX17 gene and primer design for qPCR experiments. G. Diagram of definitive endoderm (DE) differentiation with H1 XLOne-Puro-Cas13d-eGFP-U6-SOX17gRNA cells with or without dox addition. Cells were treated with CHIR99021 and Dorsomorphin in basal medium on day 0 and then cultured in basal medium suppled with 0.05% HSA and 200 μg/mL ascorbic acid for the next three days. H. Relative expression of SOX17 RNA on D4 with or without dox. I. Flow cytometry stained against SOX17 on day 4 with H1 XLOne-Puro-Cas13d-eGFP-U6-SOX17gRNA cells with or without dox addition. J-K. Quantification of Cas13d-mediated SOX17 knockdown efficiency in differentiated DE cells from H1 cell line (J) or in H9 cell line (K).