Extended Data Fig. 2: RfxCas13d is the best effector to mediate circRNA knockdown.
From: Screening for functional circular RNAs using the CRISPR–Cas13 system
a,b, Evaluation of Cas13 proteins for knocking down circRNAs (circPOLR2A and circRTN4) and their cognate mRNAs in HeLa cells revealed that RfxCas13d is the best effector for circRNA-specific KD. b, n = 63 biologically independent samples and transcript levels were normalized to ACTB. Data are presented as means values ± s.d. c, RfxCas13d/BSJ-gRNAs specifically and robustly knocked down circRNAs, but not their cognate mRNAs in HeLa cells. d, Mismatch tolerance of RfxCas13d for circRNA targeting. Guides containing single or double mismatches at varying positions across spacer sequences for circPOLR2A are shown in purple; bases flanking the BSJ site are shown in red. e, Efficiency and specificity of RfxCas13d for circRNA and cognate linear mRNA KD. Top, schematic of gRNAs targeting circPVT1 tiled 10-nt increments away from the BSJ site. Bottom, KD efficiencies of circRNA and linear RNA by each gRNA and RfxCas13d. f-g, Arrayed KD screen of 23 guides evenly tilled across BSJ of circHIPK3 (f) and circRTN4 (g). Position-effect of BSJ-gRNAs for RfxCas13d-mediated KD of circHIPK3 (f) and circRTN4 (g) at the single nucleotide level. Twenty-three guides tiled across the BSJ of circHIPK3 (f) and circRTN4 (g) are listed. KD efficiencies of circHIPK3 (f) or circRTN4 (g) and linear HIPK3 (f) or linear RTN4 (g) by each BSJ-gRNA were shown in the right. (a,c,d,e,f,g) All expression levels of RNAs were detected by qRT-PCR and were normalized to ACTB, means ± s.d. were from three independent experiments. (a,b,c) **: P < 0.01; ***: P < 0.001; ns, not significant, two-tailed student’s t-test.
