Self-Replicating RNA Vectors for Non-Integrative CRISPR Delivery
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Solution Overview
Problem
Current CRISPR gene delivery systems, such as lentiviral and retroviral systems, risk integrating the CRISPR coding sequence into the host cell genome, necessitating a robust and non-integrative method for expressing CRISPR proteins in eukaryotic cells for genome editing.
Innovation Solution
Development of synthetic, self-replicating RNA vectors based on alphaviruses that encode CRISPR proteins, which replicate without integrating into the cellular DNA, combined with guide RNAs for targeted genome editing, allowing for robust expression and precise editing without genomic integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If lentiviral or retroviral systems are used to deliver CRISPR, then robust expression of CRISPR proteins is achieved, but the risk of integration into the host cell genome increases
Solution Approach 1:
The CRISPR delivery system is segmented into separate RNA components (CRISPR RNA and guide RNA) that are delivered together but do not integrate into the host genome. The CRISPR RNA contains the coding sequence for CRISPR proteins while the guide RNA directs specificity, allowing robust expression without permanent genomic integration.
Solution Approach 2:
A self-replicating RNA intermediary is used that can replicate in the cytoplasm without integrating into nuclear DNA. This RNA intermediary serves as a temporary template for CRISPR protein production, achieving robust expression while avoiding the harmful integration effect through its non-integrative replication mechanism.
2Productivity
If viral vectors are used for CRISPR delivery, then efficient transduction is achieved, but safety concerns arise from potential insertional mutagenesis
Solution Approach 1:
The system uses transient, non-integrating RNA molecules that serve their purpose of delivering CRISPR proteins and then naturally degrade. These short-lived RNA vectors are disposable in the sense that they complete their function without permanently altering the host genome, eliminating insertional mutagenesis risk while maintaining transduction efficiency.
Solution Approach 2:
The delivery system changes its physical state from permanent genomic integration to transient cytoplasmic replication. By modifying the persistence parameter of the delivery vehicle (from stable viral DNA integration to unstable RNA replication), the system achieves efficient transduction while preventing the harmful effect of insertional mutagenesis through parameter control.
3Object-affected harmful factors
If non-integrating delivery methods are used, then safety is improved, but expression duration may be limited
Solution Approach 1:
The self-replicating RNA mechanism ensures continuous production of CRISPR proteins by repeatedly transcribing the CRISPR RNA template. This continuous action maintains robust protein expression over time without requiring permanent genomic integration, thereby extending expression duration while preserving safety.
Solution Approach 2:
The CRISPR RNA contains self-replicating elements that allow it to autonomously produce multiple copies of itself and the CRISPR proteins it encodes. This self-service capability enables prolonged expression duration without external intervention or integration, as the RNA system sustains its own production cycle within the cytoplasm.
Data Source
AI summary
Synthetic, noninfectious, self-replicating RNA vectors that encode CRISPR proteins are provided. Each self-replicating RNA vector comprises a sequence encoding a plurality of non-structural replication complex proteins from an alphavirus and a sequence encoding a CRISPR protein. Also provided are methods for genome editing in which a synthetic self-replicating RNA vector is transfected into cells along with at least one corresponding guide RNA.