Modified CRISPR Guide RNA for Nuclease Stability
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Solution Overview
Problem
Current methods for delivering guide RNA in CRISPR-Cas systems face challenges such as triggering an innate immune response, rapid degradation by nucleases, and uncontrollable transcription, leading to toxicity and instability in mammalian cells.
Innovation Solution
Development of modified RNA oligonucleotides with 2′-O-alkyl and 2′-O-fluoro modifications, as well as end-modifications like inverted-dT and phosphorothioate linkages, to enhance stability and reduce immunogenicity, allowing for effective use in CRISPR-Cas systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If native guide RNA is used in CRISPR-Cas systems, then the system can function for genome editing, but the RNA triggers an innate immune response and is rapidly degraded by nucleases
Solution Approach 1:
The patent applies chemical modifications to the guide RNA structure, specifically 2′-O-alkyl and 2′-O-fluoro modifications at selected nucleotide positions, as well as end-modifications like inverted-dT and phosphorothioate linkages. These parameter changes in the RNA's chemical structure enhance stability against nucleases and reduce immunogenicity while preserving CRISPR-Cas system functionality.
Solution Approach 2:
The patent creates composite RNA structures by combining modified nucleotides with unmodified sequences. The guide RNA comprises a mix of chemically modified and unmodified nucleotides, where the modified portions provide stability and reduced immunogenicity while the unmodified portions maintain binding affinity and Cas9 recruitment capability.
2Ease of manufacture
If unmodified RNA is transfected into mammalian cells, then delivery is straightforward, but the RNA is quickly degraded and causes toxicity
Solution Approach 1:
The patent modifies the chemical parameters of the RNA molecule by introducing 2′-O-alkyl and 2′-O-fluoro modifications that protect against nuclease degradation. These modifications change the RNA's physical and chemical properties to enhance stability in mammalian cells without compromising deliverability through standard transfection methods.
Solution Approach 2:
The patent applies protective chemical modifications beforehand to the guide RNA to cushion it against degradation by nucleases and immune recognition. The phosphorothioate linkages and 2′-O-alkyl modifications act as preemptive protective measures that prevent rapid degradation and reduce toxicity upon cellular uptake.
3Quantity of substance
If endogenous transcription is used to produce guide RNA, then continuous supply is achieved, but transcription becomes uncontrollable leading to toxicity
Solution Approach 1:
The patent employs synthetic guide RNA with chemical modifications that provides transient, controlled activity. The modified RNA is designed to be stable enough to persist long enough to achieve gene editing but ultimately degraded, avoiding the accumulation and toxicity associated with endogenous transcription. This disposable approach allows precise control over RNA levels and duration of action.
Data Source
AI summary
This invention pertains to modified compositions for use in CRISPR systems, and their methods of use. In particular, length-modified and chemically-modified forms of crRNA and tracrRNA are described for use as a reconstituted guide RNA for interaction with Cas9 of CRISPR systems. The resultant length-modified and chemically-modified forms of crRNA and tracrRNA are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRISPR Cas9 endonuclease system.