Guide RNA-Cas Protein Conjugates for Precision Genome Editing
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Solution Overview
Problem
Current CRISPR-Cas systems face challenges such as guide-independent off-target activities, lack of single nucleotide-resolution in editing, and difficulties in viral delivery due to size constraints of delivery vehicles like AAVs.
Innovation Solution
The development of conjugates of guide RNA(s)-Cas protein (RNP) complexes, where NLS sequences are added to guide RNA conjugates instead of proteins, and the use of synthetic guide RNA-DNA template conjugates in STAR editors for precise genome and epigenome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If NLS sequences are added to guide RNA conjugates instead of proteins, then guide-independent off-target activities are prevented and controlled gene manipulations are enabled, but the complexity of guide RNA conjugate design increases
Solution Approach 1:
The invention segments the targeting function by adding NLS sequences specifically to guide RNA conjugates rather than to the entire protein complex. This allows the guide RNA to be independently modified with nuclear localization signals, separating the targeting mechanism from the Cas protein and enabling controlled dosing of guide RNA-peptide conjugates to prevent off-target activities.
Solution Approach 2:
Instead of adding NLS sequences to the protein component (Cas protein), the invention inverts the approach by adding NLS sequences to the guide RNA component. This inversion allows for controlled gene manipulations through controlled dosing of guide RNA-peptide conjugates and prevents guide-independent off-target activities.
2Manufacturing precision
If conjugates of guide RNA-Cas protein complexes are used for precise gene editing, then editing precision is enhanced and off-target effects are reduced, but the size and complexity of delivery vehicles increases
Solution Approach 1:
The invention segments the delivery problem by using non-viral delivery methods for guide RNA conjugates and reserving viral vectors (AAV) only for delivering the Cas protein. This division allows precise gene editing through guide RNA-Cas protein conjugates while avoiding the size constraints of viral delivery vehicles for the entire complex.
Solution Approach 2:
The invention uses guide RNA conjugates as intermediaries that can be delivered non-virally to cells, where they then form functional complexes with Cas protein (delivered via AAV or other means). This intermediary approach enables precise editing without requiring the entire complex to be packaged in viral vectors.
3Reliability
If chemical modifications are made to guide RNA to increase stability and enhance specificity, then toxicities are minimized and target recognition is improved, but the complexity of guide RNA synthesis and modification increases
Solution Approach 1:
The invention applies parameter changes by introducing specific chemical modifications to guide RNA at defined positions (e.g., 2'-fluoro, 2'-methoxy, LNA modifications) to optimize stability and specificity. These parameter changes in the chemical structure of guide RNA enable improved target recognition and reduced toxicities while maintaining manufacturability through established synthesis methods.
Data Source
AI summary
Provided herein are compositions of conjugates of a guide RNA(s)-CRISPR Cas protein (RNP) complex. The conjugate comprises a guide RNA(s)-CRISPR Cas protein (RNP) complex and one or more molecules selected from PEG, non-PEG polymers, ligands for cellular receptors, lipids, oligonucleotides, polysaccharides and peptides and chemically linked to the Cas protein and/or guide RNA(s). Also provided a guide RNA-DNA template conjugate comprises a DNA template with 5-methylcytosines at specific positions for epigenome editing by DNA methylation at a single nucleotide resolution level. The conjugates are delivered to targeted cells as RNP complexes, or formed in targeted cells from guide RNA conjugates and an mRNA or a viral vector encoding a Cas protein, or formed in targeted cells from a crRNA conjugate(s) and a viral vector encoding both a Cas protein and a tracrRNA. Also provided are preparation methods and uses of these conjugates.