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. 2020 Oct 30;49(3):1330–1344. doi: 10.1093/nar/gkaa865

Figure 4.

Figure 4.

Flap-type aptamers had efficient inhibitory activities for Cas9 in human cells. (A) Simultaneous delivery of Cas9 RNP and an aptamer markedly interfered with Cas9-mediated gene editing in human cells. Chromosomally integrated Scarlet-293 stable cells were transfected with Cas9 RNP, as well as aptamers (2 h before [B2], simultaneously with [S], or 2 h after [A2] Cas9 RNP transfection). At 72 h post-transfection, flow cytometry was performed to quantify the indel frequencies based on lost Scarlet expression in Scarlet-293 cells. Data are presented as means ± S.D. from three independent experiments. (B) An LNA-modified aptamer for Cas9 RNP markedly inhibited genome editing compared with a normal DNA aptamer at 200 nM. (C)The LNA-modified aptamer interfered with Cas9-mediated gene editing in a dose-dependent manner. The LNA-modified aptamer (50 nM) completely blocked gene editing (Cas9: crRNA/tracrRNA: inhibitor molar ratio, 1:1:1.4). LNA-modified nucleotides are indicated by yellow. Underlining indicates nucleotides with a phosphorothioate backbone. (D) LNA-modified aptamers could turn off Cas9-mediated genome editing for endogenous targets in HEK293FT cells. Indels caused by Cas9 RNP were detected in the T7E1 assay. Cells that were not transfected with Cas9/crRNA/tracrRNA and the aptamer were used as a negative control. Digested bands are indicated with star symbols. (E) Cpf1 editing of GFP1 was blocked in vitro in the presence of the aptamer designed for Cpf1. Since Cpf1 recognizes the PAM sequence (yellow) at the 3′ side of crRNA, a flap-type aptamer designed for Cpf1 is needed for the flap at the 5′ end of the aptamer to form a duplex with crRNA. Three mutants with a G:T mismatch in the PAM region showed decreased inhibitory activities. Schematic illustration of the interaction between crRNA (blue) and the aptamer designed for Cpf1 (purple) at the GFP1 site, with complementary base pairing indicated in red. (F) The inhibitory activities of Cpf1 aptamers were confirmed at three target sites in EGFR in vitro.