Modified Guide RNA for CRISPR Nuclease Resistance
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Solution Overview
Problem
Current methods for delivering guide RNAs 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
The use of modified RNA oligonucleotides, including 2′-O-alkyl and 2′-O-fluoro modified RNA, LNA or BNA modified RNA, and end-modifications like inverted-dT base or propanediol group to enhance nuclease resistance and stability, allowing for reduced toxicity and extended functionality in CRISPR-Cas systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If unmodified guide RNAs are used in CRISPR-Cas systems, then the system can function with native sequences, but the guide RNAs are rapidly degraded by nucleases and trigger innate immune responses
Solution Approach 1:
The patent applies chemical modifications to the guide RNA backbone, specifically 2′-O-alkyl and 2′-O-fluoro modifications, which change the chemical parameters of the RNA molecule to increase resistance to nuclease degradation while maintaining CRISPR-Cas system functionality
Solution Approach 2:
The patent creates composite RNA structures by combining modified nucleotide building blocks (containing 2′-O-alkyl or 2′-O-fluoro modifications) with standard nucleotide sequences to form guide RNAs that possess both the stability of modified RNA and the sequence-specific functionality of native guide RNAs
2Productivity
If high dosages of guide RNA are used to ensure sufficient activity, then gene editing efficiency improves, but toxicity increases due to immune activation
Solution Approach 1:
The chemical modifications to the guide RNA backbone change the immunogenicity parameters of the RNA, reducing its ability to trigger innate immune responses while maintaining or enhancing its gene editing activity, thereby allowing effective dosages to be reduced
3Reliability
If native crRNA:tracrRNA complexes are used, then the CRISPR-Cas system functions as designed in bacteria, but delivery and control in mammalian cells becomes difficult
Solution Approach 1:
The patent merges the crRNA and tracrRNA sequences into a single chimeric guide RNA molecule, simplifying delivery requirements and eliminating the need for separate transcription control of two RNA components while maintaining the functional complex formation with Cas9
Solution Approach 2:
The patent introduces chemical modifications to the chimeric guide RNA that enhance its stability and reduce immunogenicity, improving the ease of operation in mammalian cell systems while preserving the essential CRISPR-Cas9 cleavage functionality
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 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 are economical to produce and can be tailored to have unique properties relevant to their biochemical and biological activity in the context of the CRIPSR Cas9 endonuclease system.