Optimized mRNA Encoding Cas9 for Genome Editing
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Solution Overview
Problem
Current methods for delivering CRISPR/Cas9 system components, such as Cas9, to target cells face challenges including vector integration, limited stability of RNP complexes, and immune responses induced by mRNA.
Innovation Solution
The use of optimized messenger RNAs (mRNAs) encoding a site-directed endonuclease like SpCas9, combined with guide RNAs, which are formulated in lipid nanoparticles to enhance delivery and activity while minimizing immune activation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If plasmid or viral vectors are used for Cas9 delivery, then delivery efficiency is improved, but risk of vector integration into the genome increases
Solution Approach 1:
The patent uses mRNA as a transient, non-integrating delivery vehicle that provides temporary Cas9 expression without permanent genomic integration. The mRNA is degraded after serving its purpose, eliminating the risk of vector integration while maintaining delivery efficiency.
Solution Approach 2:
The patent introduces lipid nanoparticles as an intermediary delivery system that transports mRNA into cells without requiring viral vectors or plasmid integration. This intermediary approach achieves efficient delivery while avoiding direct genomic integration risks.
2Speed
If recombinant Cas9 polypeptide complexed to gRNA (RNP complexes) is used for delivery, then delivery speed is improved, but stability of complexes in cells or plasma is limited
Solution Approach 1:
The patent performs preliminary action by transcribing Cas9 mRNA and delivering it to cells before the actual genome editing occurs. The mRNA is processed and translated into Cas9 protein within the cell, ensuring the enzyme is ready when needed while maintaining stability throughout the process.
Solution Approach 2:
The patent uses mRNA as an intermediary between the stable Cas9 gene sequence and the active Cas9 protein. This intermediary approach allows for stable storage and delivery of the genetic information while producing the active enzyme at the appropriate time and location within the cell.
3Adaptability or versatility
If mRNA expressing Cas9 is used for delivery, then expression flexibility is improved, but innate immune responses are induced reducing Cas9 expression
Solution Approach 1:
The patent applies parameter changes by chemically modifying nucleosides within the mRNA sequence (e.g., replacing uridine with pseudouridine or 5-methoxyuridine). These parameter changes in the molecular structure reduce immune recognition while maintaining translation efficiency and Cas9 expression flexibility.
Solution Approach 2:
The patent converts the potential harm of immune recognition into a benefit by using chemical modifications that specifically prevent immune detection. The modifications maintain the biological functionality of mRNA while eliminating the harmful immune response, effectively turning a vulnerability into an advantage.
Data Source
AI summary
The present disclosure provides optimized mRNAs encoding a site-directed endonuclease for use in a CRISPR/Cas system. Also provided herein are delivery systems for use of the CRISPR/Cas system in methods of in vivo and ex vivo genome editing.