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. 2020 Dec 10;11:594576. doi: 10.3389/fgene.2020.594576

FIGURE 1.

FIGURE 1

Tracking the expanded CUG RNA foci and stress granules in COS-M6 cells. (A) Mammalian expression vector pX330 encoding crRNA under a pU6 promoter and dLshCas13a (LshCas13aR1278A-eGFP) under a pCMV promoter. The crRNA contains the direct repeat hairpin (DR, gray box) and the spacer (black box). The dLshCas13a is flanked by an N-terminal nucleoplasmin NLS and a C-terminal c-Myc NLS (Ray et al., 2015). (B) Structure of the crDM1 with DR sequence highlighted in gray, spacer in black and target CUG repeats in blue. The 3′-PFS uracil is highlighted in red. (C) Without the expression of crRNA, dLshCas13a primarily localizes in the nucleus. (D) Comparison of non-targeting (crλNT) and CUG-targeting (crDM1) dLshCas13a tracking nuclear CUG foci in COS-M6 cells. The expression of the interrupted CUGx960 repeats is driven by a CMV promoter in DT960 plasmid. (E) Comparison of non-targeting (crλNT) and ACTB-targeting (crACTB) dLshCas13a tracking cytoplasmic stress granules in COS-M6 cells. The stress condition was induced by adding 200 μM sodium arsenite for 1 h 30 min before fixation. Stress granules are labeled with anti-G3BP1 Alexa 594 antibodies by immunofluorescence (IF).