Journal: Molecular Therapy. Nucleic Acids

Article Title: Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease

doi: 10.1016/j.omtn.2025.102789

Figure Lengend Snippet: Evaluation of CASFx to modulate splicing defects of ABCA4 c.5461-10T>C in ARPE19 (A) A schematic illustration of the pSPL3–ABCA4 c.5461-10T>C minigene and the CRISPR splicing factors CASFx-1 (RBFOX1N-dCasRx-C) and CASFx-3 (dCasRx-RBM38), as well as two sgRNA target sites downstream of exon 39 (E39) and exon 40 (E40), respectively (green). ABCA4 exons (orange), and flanking introns (gray) were cloned into the pSPL3 vector (black) along with the mutation between two pSPL3 exons; splice donor vector (SDv, green) and splice acceptor vector (SAv, blue). The ABCA4 splice variants were amplified using the SD6 and SA2 primers. (B) Expected splicing products from the ABCA4 c.5461-10T>C minigene. Normal splicing of the minigene gives rise to an FL product containing both exon 39 and 40 (514 bp) while skipping of exon 39 or exon 39 and 40 generates shorter isoforms (390 and 260 bp). (C) RT-PCR results of ABCA4 splice variants in ARPE19 cells transfected with the ABCA4 minigene system (MT), CASFx-1, CASFx-3, and specified sgRNAs. ACTB was used as a housekeeping gene. Mock control represents ARPE19 without transfection of the ABCA4 minigene system. (D) Gel quantification of relative transcript ratio of ABCA4 splice variants compared to MT control without treatment. Relative expression levels compared to MT control without treatment are shown. Data are represented as mean ± SEM ( n = 3 for CASFx-3 ex39 sgRNA1+2 ex40 sgRNA 1 + 2, n = 2 for the rest). ∗: p < 0.05. (E) qPCR quantification of the relative expression level of ABCA4 splice variants compared to MT control without treatment, and (F) relative expression level of disease transcript to FL transcript. Data are presented as mean ± SEM ( n = 3). ∗∗: p < 0.01.

Article Snippet: CASFx-1 (RBFOX1N-dCasRx-C) and CASFx-3 (dCasRx-RBM38) were obtained from Addgene (Plasmid #118635 and #118638).

Techniques: CRISPR, Clone Assay, Plasmid Preparation, Mutagenesis, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, Control, Expressing

Journal: Molecular Therapy. Nucleic Acids

Article Title: Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to target ABCA4 splice variants in Stargardt disease

doi: 10.1016/j.omtn.2025.102789

Figure Lengend Snippet: Evaluation of CASFx to modulate splicing defects of ABCA4 c.4773+3A>G in ARPE19 (A) A schematic illustration of the pSPL3– ABCA4 c.4773+3A>G minigene and the CRISPR splicing factors CASFx-1 (RBFOX1N-dCasRx-C) and CASFx-3 (dCasRx-RBM38), as well as two sgRNA target sites downstream of exon 33 (E33) (green). Positions of SD6 and SA2 primers for splicing product amplification are displayed. (B) Expected splicing products from the ABCA4 c.4773+3A>G minigene. Normal splicing of the minigene gives rise to an FL product containing both exon 33 and 34 (441 bp) while skipping of exon 33 or exon 33 and 34 generates shorter isoforms (335 and 260 bp). (C) RT-PCR results of ABCA4 splice variants in ARPE19 cells transfected with the ABCA4 minigene system (MT), CASFx-1, CASFx-3 and specified sgRNAs. ACTB was used as a housekeeping gene. Mock control represents ARPE19 without transfection of the ABCA4 minigene system. (D) Gel quantification of relative transcript ratio of ABCA4 splice variants compared to MT control without treatment, and (E) ratio of disease transcript to FL transcript. Data are represented as mean ± SEM ( n = 2).

Article Snippet: CASFx-1 (RBFOX1N-dCasRx-C) and CASFx-3 (dCasRx-RBM38) were obtained from Addgene (Plasmid #118635 and #118638).

Techniques: CRISPR, Amplification, Reverse Transcription Polymerase Chain Reaction, Transfection, Control