Fig. 4

Specific circRNA KO with base changes at back-splice sites of predominantly back-spliced exons by BE. A Schematic of circRNA knockout by BE-mediated back-splice sites mutation. BEs could introduce base changes at back-splice sites of predominantly back-spliced novel exons (pink bar), resulting in corresponding circRNA knockout with little effect on cognate linear RNA expression. B Base changes at 3′ bss of the novel exon in the RALY gene locus by hA3A-eBE-Y130F repressed the back-splice for circRALY-nov. Top, Schematic of partial RALY gene organization. Back-spliced novel exon was highlighted by pink bar. Context sequences of targeted 3′ (b)ss were shown by a, t, c, and g for intron or by A, T, C, and G for exon; Middle, G-to-A base change ratio at targeted 3′ (b)ss of predominantly back-spliced novel exon in the RALY gene locus; Bottom, evaluation of back-splice and splice changes by RT-qPCR using primers labeled on the top. Since the identified novel exon was only back-spliced in circRALY-nov, base changes at its 3′ bss only affect back-splice of circRALY-nov, but not back-splice for canonical splice for linear RALY RNA(s) with annotated exons. Error bar represents SD from three independent replicates. ns, not significant; ∗∗∗, P < 0.001; Student’s t test. C Base changes at 3′ bss of the novel exon in the CAMK1D gene locus by hA3A-eBE-Y130F repressed the back-splice for circCAMK1D-nov. Refer to B for details