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. 2022 Jan 10;23:16. doi: 10.1186/s13059-021-02563-0

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© The Author(s) 2022

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 3 — Achievement of CDR1as/ciRS-7 knockout by base editing at its 5′ back-splice site. A Schematic of the CDR1as/ciRS-7 gene organization and mapped RNA-seq signals in 293FT cells. Top, multiple transcripts were predicted/reported in the CDR1as/ciRS-7 gene locus, including a long noncoding RNA containing CDR1as/ciRS-7-residing exon (blue bar) and a cryptic exon (blank bar). Bottom, the circular molecule, CDR1as/ciRS-7, was confirmed as the major transcript produced from its gene locus, enriched after RNase R treatment. B Design of base changes at 5′ (b)ss of CDR1as/ciRS-7 by hA3A-eBE-Y130F. Top, schematic of partial CDR1as/ciRS-7 gene organization. The back-spliced CDR1as/ciRS-7 (blue bar) and a cryptic exon (blank bar) were reported to be also spliced in a long noncoding RNA. Middle, primers for RT-qPCR and probes for northern blotting. Bottom, context sequences of targeted 5′ (b)ss were shown by a, t, c, and g for intron or by A, T, C, and G for exon; G-to-A base change ratio at targeted 5′ (b)ss of back-spliced CDR1as/ciRS-7 exon was examined in transfected 293FT cell mixture. C Repression of CDR1as/ciRS-7 back-splice by base changes at its 5′ bss. RT-qPCR was performed with primers labeled in B. D Selection of monoclones with corresponding base editing changes at the 5′ bss of CDR1as/ciRS-7. Four monoclones were identified with almost 100% G-to-A base change at the exon boundary of the CDR1as/ciRS-7 5′ (b)ss, and among them, monoclones #2 and #4 have an additional G-to-A change (~ 67%) at the intron boundary of the CDR1as/ciRS-7 5′ bss. Four monoclones with unchanged bases at the 5′ bss of CDR1as/ciRS-7 were used as controls (#1 is showed in this panel). E Expression of CDR1as/ciRS-7 was undetected in the four selected monoclones with base editing changes at the 5′ bss of CDR1as/ciRS-7, revealed by northern blotting with two probes (1–157 nt and 1–1485 nt). Total RNAs were denatured and then resolved on 1.5% native agarose gel. F Back-splice of CDR1as/ciRS-7 was barely detected in the four selected monoclones with base editing changes at the 5′ bss of CDR1as/ciRS-7, revealed by RT-qPCR. Canonical splice along its cognate linear RNA was further compared by parallel RT-qPCR. n.d. indicates non-detected. C, F Error bar represents SD from three independent replicates. ∗∗∗, P < 0.001, Student’s t test