CRISPR-dCas13d-based deep screening of proximal and distal splicing-regulatory elements.
Nat Commun
; 15(1): 3839, 2024 May 07.
Article
in En
| MEDLINE
| ID: mdl-38714659
ABSTRACT
Pre-mRNA splicing, a key process in gene expression, can be therapeutically modulated using various drug modalities, including antisense oligonucleotides (ASOs). However, determining promising targets is hampered by the challenge of systematically mapping splicing-regulatory elements (SREs) in their native sequence context. Here, we use the catalytically inactive CRISPR-RfxCas13d RNA-targeting system (dCas13d/gRNA) as a programmable platform to bind SREs and modulate splicing by competing against endogenous splicing factors. SpliceRUSH, a high-throughput screening method, was developed to map SREs in any gene of interest using a lentivirus gRNA library that tiles the genetic region, including distal intronic sequences. When applied to SMN2, a therapeutic target for spinal muscular atrophy, SpliceRUSH robustly identifies not only known SREs but also a previously unknown distal intronic SRE, which can be targeted to alter exon 7 splicing using either dCas13d/gRNA or ASOs. This technology enables a deeper understanding of splicing regulation with applications for RNA-based drug discovery.
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Introns
/
RNA Splicing
/
Exons
/
Survival of Motor Neuron 2 Protein
/
CRISPR-Cas Systems
/
RNA, Guide, CRISPR-Cas Systems
Limits:
Humans
Language:
En
Journal:
Nat Commun
Journal subject:
BIOLOGIA
/
CIENCIA
Year:
2024
Document type:
Article
Affiliation country: