Fig. 5: CAAA tract is indispensable for seRNA-1 function.

a Schematic illustration of CAAA deletion medicated by CRISPR-Cas9 editing in C2C12 cells. Top: black box depicts the deletion region. Primer set 1 (1F and 1R) was used for cDNA genotyping. Primer set 2 (2F and 2R) was used for qRT-PCR analysis. Middle: CAAA tract sequence is highlighted in red. sgRNAs were designed to delete the underlined sequence encompassing the CAAA tract. Bottom: Result from Sanger sequencing confirmed the CAAA deletion. b Biotinylated seRNA-1 transcripts of full length (FL) or 5′ end fragment with or without CAAA deletion (ΔCAAA) were used in RNA pull-down assay to reveal seRNA-1 binds hnRNPL through the CAAA tract embedded in its 5′ region. c hnRNPL RIP was performed in WT or the generated CAAA deletion (KO) cells to show hnRNPL/seRNA-1 binding was abolished in the KO cells as compared to WT control cells. Enrichment was determined as RNA associated to hnRNPL IP relative to IgG control. d Expression of the associated target genes, Mb and Apol6, was decreased but seRNA-1 was increased in the KO cells as measured by qRT-PCR. e Schematic illustration of the CRISPR-Cas9 mediated in vivo deletion of CAAA tract. Pax7^Cas9 mouse at postnatal 10 (P10) age was intramuscularly (IM) injected with 5 × 10¹¹ vg AAV9-sgRNAs viruses and the infected muscles were recovered for analysis at 4 weeks later (P38). n = 3 per group. f Detection of CAAA excision by genomic PCR in muscle tissues injected with AAV9-sg-seRNA-1, compared to AAV-sg-Control. Un-edited product, 304 bp; deletion product (red asterisk), 220 bp. g qRT-PCR was performed to measure the levels of seRNA-1, Mb and Apol6 in the above injected muscles. n = 3 per group. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t-test (c, d, g), *P < 0.05, **P < 0.01, and ***P < 0.001. Source data are provided as a Source Data file.