RNA-Guided Trans-Splicing for Pre-mRNA Mutation Correction
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Solution Overview
Problem
Current technologies lack robust systems for sequence-specific modifications at the pre-mRNA level, particularly for correcting deleterious mutations and enhancing gene expression, which are not efficiently addressed by existing RNA targeting methods.
Innovation Solution
The development of engineered compositions comprising a catalytically inactive RNA-binding Cas polypeptide (dCas) and a trans-splicing donor construct, which includes a guide sequence, an intron, a splice acceptor, a donor RNA, and a polyA tail, enabling sequence-specific binding and splicing of heterologous sequences into target pre-mRNA to correct mutations or enhance gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If existing RNA targeting methods are used, then RNA can be targeted, but sequence-specific modifications at the pre-mRNA level cannot be efficiently achieved
Solution Approach 1:
The patent uses a catalytically inactive Cas13 (dCas13) as an intermediary RNA-binding protein that directs the trans-splicing donor construct to the target pre-mRNA sequence. The guide RNA acts as another intermediary that mediates specific binding between dCas13 and the target sequence, enabling precise localization without catalytic activity. This intermediary approach resolves the contradiction by providing sequence-specific targeting while allowing efficient trans-splicing modification.
Solution Approach 2:
The invention segments the modification system into distinct functional components: dCas13 for RNA binding, guide RNA for sequence specificity, trans-splicing donor construct for modification, and intron/splice acceptor elements for efficient splicing. This segmentation allows each component to be optimized independently, achieving both high precision targeting and efficient modification productivity.
2Productivity
If trans-splicing donor construct is designed with intron and splice acceptor, then splicing efficiency is improved, but construct complexity increases
Solution Approach 1:
The trans-splicing donor construct contains self-contained splicing signals (intron with splice donor and splice acceptor sites) that enable the construct to undergo splicing autonomously when bound to the target pre-mRNA. The spliceosome machinery naturally recognizes these signals and performs the splicing reaction without requiring additional engineering or complex control mechanisms, thus improving efficiency while managing complexity.
3Measurement precision
If catalytically inactive dCas is used for RNA binding, then sequence-specific binding is achieved, but catalytic RNA modification activity is lost
Solution Approach 1:
The patent introduces a trans-splicing donor construct as an intermediary that carries the modification capability separately from the binding function. The dCas13-provides binding specificity, while the donor construct with its intron and splice acceptor provides the modification capability. When the dCas13-guide complex binds the target pre-mRNA, it recruits the splicing machinery to perform the modification, thus separating binding precision from modification capability.
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
This approach allows for precise modification of pre-mRNA by facilitating the trans-splicing of donor RNA into target pre-mRNA, correcting mutations and enhancing gene expression, thereby providing a robust method for modifying endogenous mRNA and increasing the expression of therapeutic or functional proteins.
Implementation Method 1
the guide is capable of forming a complex with the dCas and directing sequence-specific binding of the complex to a target pre-cursor mRNA
Implementation Method 2
facilitating splicing of the exon and the heterologous donor RNA into the target pre-mRNA to generate a modified mRNA
Data Source
AI summary
Recent advances in the understanding of two CRISPR-Cas systems, namely, Type VI (Cas13a-d) and Type III (Type III-A-B), have shown both of them to have the ability to efficiently target RNA. Provided herein are compositions and methods for trans-splicing precursor mRNA using a catalytically-inactive Cas protein (dCas) and a trans-splicing construct comprising a guide RNA, an intron, a splice acceptor, donor RNA, and a poly A tail. The dCas is capable of forming a complex with the guide RNA and directing sequence-specific binding of the complex to a target precursor mRNA for genetic modification and any polypeptides derived thereof. The technology has important potential therapeutic applications such as correcting genetic mutations through exon replacement, insertion of transgenes, and increasing gene expression, and in non-therapeutic applications such as cell- and tissue-specific diagnostics.