PT - JOURNAL ARTICLE AU - Liou, Roxanne Hsiang-Chi AU - Urrutia-Cabrera, Daniel AU - Westin, Ida Maria AU - Golovleva, Irina AU - Liu, Guei-Sheung AU - Kumar, Satheesh AU - McLenachan, Samuel AU - Chen, Fred Kuanfu AU - Hsu, Fei-Ting AU - Edwards, Tom AU - Martin, Keith R AU - Cheng, Albert Wu AU - Wong, Raymond C.B. TI - Using RNA-targeting CRISPR-Cas13 and engineered U1 systems to reduce <em>ABCA4</em> splice variants in Stargardt disease AID - 10.1101/2024.03.08.584155 DP - 2024 Jan 01 TA - bioRxiv PG - 2024.03.08.584155 4099 - http://biorxiv.org/content/early/2024/03/09/2024.03.08.584155.short 4100 - http://biorxiv.org/content/early/2024/03/09/2024.03.08.584155.full AB - Dysregulation of the alternative splicing process results in aberrant mRNA transcripts, leading to dysfunctional proteins or nonsense-mediated decay that cause a wide range of mis-splicing diseases. Development of therapeutic strategies to target the alternative splicing process could potentially shift the mRNA splicing from disease isoforms to a normal isoform and restore functional protein. As a proof of concept, we focus on Stargardt disease (STGD1), an autosomal recessive inherited retinal disease caused by biallelic genetic variants in the ABCA4 gene. The splicing variants c.5461-10T&gt;C and c.4773+3A&gt;G in ABCA4 cause the skipping of exon 39-40 and exon 33-34 respectively. In this study, we compared the efficacy of different RNA-targeting systems to modulate these ABCA4 splicing defects, including four CRISPR-Cas13 systems (CASFx-1, CASFx-3, RBFOX1N-dCas13e-C and RBFOX1N-dPspCas13b-C) as well as an engineered U1 system (ExSpeU1). Using a minigene system containing ABCA4 variants in the human retinal pigment epithelium ARPE19, our results show that RBFOX1N-dPspCas13b-C is the best performing CRISPR-Cas system, which enabled up to 80% reduction of the mis-spliced ABCA4 c.5461-10T&gt;C variants and up to 78% reduction of the ABCA4 c.4773+3A&gt;G variants. In comparison, delivery of a single ExSpeU1 was able to effectively reduce the mis-spliced ABCA4 c.4773+3A&gt;G variants by up to 84%. We observed that the effectiveness of CRISPR-based and U1 splicing regulation is strongly dependent on the sgRNA/snRNA targeting sequences, highlighting that optimal sgRNA/snRNA designing is crucial for efficient targeting of mis-spliced transcripts. Overall, our study demonstrated the potential of using RNA-targeting CRISPR-Cas technology and engineered U1 to reduce mis-spliced transcripts for ABCA4, providing an important step to advance the development of gene therapy to treat STGD1.Competing Interest StatementThe authors have declared no competing interest.