RNA-Guided dCas13-RBM25 Fusion for Endogenous Exon Modulation
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Solution Overview
Problem
Current methods for modulating alternative splicing, such as antisense oligonucleotides (ASOs) and CRISPR-Cas systems, are inefficient, lack specificity, and are not scalable for high-throughput interrogation of exon function, particularly in activating endogenous exons.
Innovation Solution
A fusion protein comprising a catalytically deactivated Cas13d (dCas13d) and the splicing factor RBM25, guided by one or more RNAs targeting an intron downstream of an exon, efficiently activates or represses splicing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If antisense oligonucleotides (ASOs) are used to modulate alternative splicing, then splicing repression can be achieved, but the effect is transient and requires laborious tiling experiments
Solution Approach 1:
The patent replaces the chemical mechanism of ASOs with a CRISPR-based molecular system. The dCas13-RBM25 fusion protein guided by gRNAs provides a programmable, sequence-specific mechanism for splicing modulation that can be rapidly redesigned without laborious tiling experiments, thereby improving productivity while maintaining reliability
Solution Approach 2:
The patent changes the fundamental parameter of splicing modulation from transient chemical binding (ASOs) to sustained protein-RNA complex formation. The dCas13-RBM25 fusion creates a stable ribonucleoprotein complex that provides durable splicing effects, eliminating the transient nature of ASO-mediated modulation
2Reliability
If typical ASOs are used for splicing activation, then exon inclusion can be promoted, but it requires a proximal repressor element that limits generalizability
Solution Approach 1:
The dCas13-RBM25 fusion protein serves multiple functions: it can activate exons by recruiting splicing activators, repress exons by blocking splice sites, and does so through a universal mechanism that does not require proximal repressor elements. This multi-functionality and lack of contextual requirements provide broad targeting generalizability across different exon contexts
Solution Approach 2:
The patent introduces RBM25 as an intermediary splicing factor that mediates both activation and repression functions. This intermediary protein can be recruited to different locations and contexts to achieve diverse splicing outcomes, providing versatility without requiring context-specific proximal elements
3Reliability
If CRISPR-Cas nucleases are used for exon deletion, then exon removal can be achieved, but activation capability is lost
Solution Approach 1:
Instead of using nucleases to remove exons (deletion approach), the patent inverts the strategy by using a catalytically deactivated Cas13d protein that binds to pre-mRNA and modulates splicing through steric blocking and recruitment of splicing factors. This inversion enables both activation and repression without permanent genomic changes, maintaining versatility while improving reliability through precise RNA-targeting
4Reliability
If dCasRx is fused to splicing factors for exon activation, then activation can be achieved, but multiple gRNAs are required and efficiency is reduced
Solution Approach 1:
The patent optimizes the gRNA targeting parameter by designing guides that target specific locations within introns or exons to maximize activation efficiency. By carefully selecting target positions and optimizing gRNA sequences, the system achieves effective exon activation with fewer gRNAs, reducing delivery complexity while maintaining high reliability
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
dCasRx-RBM25 achieves robust and specific modulation of endogenous exons with a single guide RNA, outperforming other splicing factors, and demonstrates versatile exon activation in various contexts.
Implementation Method 1
a fusion protein comprising a catalytically deactivated Cas13d (dCas13d) and a splicing factor, wherein the splicing factor is RBM25 polypeptide
Implementation Method 2
The recent discovery and engineering of RNA-targeting type-VI CRISPR systems have greatly advanced possibilities for directing specific transcriptome perturbations
Implementation Method 3
one or more guide RNAs, wherein the one or more guide RNAs targets an intron downstream of an exon of a first pre-mRNA
Implementation Method 4
Alternative splicing is a process that allows a single pre-mRNA to produce multiple isoforms by selective usage of splice sites
Data Source
AI summary
The present disclosure provides fusion proteins comprising a catalytically deactivated Cas13d and the splicing factor RBM25. The present disclosure also provides isolated nucleic acids encoding the fusion proteins and cells comprising said nucleic acids. The present disclosure further provides methods for modulating alternative splicing.