NLS-Conjugated gRNA for CRISPR Nuclear Delivery
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Solution Overview
Problem
The degradation of guide RNA (gRNA) by nucleases and inefficient assembly and transport within cells pose challenges for effective gene editing in CRISPR/Cas systems, limiting the persistence and functionality of gRNA in therapeutic applications.
Innovation Solution
Conjugating a nuclear localization signal (NLS) to the gRNA through a linker, particularly with a cysteine residue, enhances its potency by improving trafficking to the nucleus and protecting it from cytosolic RNases, thereby increasing the local concentration and stability of the gRNA for effective ribonucleoprotein formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gRNA is used without NLS conjugation, then the structure is simpler and easier to manufacture, but the gRNA is degraded by nucleases and cannot efficiently reach the nucleus
Solution Approach 1:
The patent combines the gRNA molecule with an NLS peptide sequence through chemical conjugation, creating a hybrid molecule that merges the functions of nuclear targeting (NLS) and guide RNA activity. This resolves the contradiction by integrating the protective and delivery functions directly into the gRNA structure, preventing nuclease degradation and ensuring nuclear delivery without requiring separate delivery mechanisms.
Solution Approach 2:
The invention creates a composite molecular structure consisting of RNA nucleotides covalently linked to amino acid residues of the NLS peptide. This composite gRNA-NLS conjugate combines the properties of both components: the RNA provides guide function while the peptide provides nuclear localization and protection from degradation, thereby improving reliability without excessive complexity.
2Reliability
If gRNA is modified with multiple modifications to enhance stability, then the gRNA resistance to nucleases improves, but the assembly efficiency into RNP and transport to nucleus decreases
Solution Approach 1:
Instead of applying multiple separate modifications to gRNA, the patent merges the protective function into a single NLS peptide conjugation. The NLS peptide provides both nuclease protection and nuclear targeting in one modification, avoiding the cumulative negative effects of multiple modifications on RNP assembly and transport efficiency.
3Reliability
If gRNA is conjugated to NLS sequence, then the trafficking to nucleus and protection from RNases is improved, but the manufacturing process becomes more complex
Solution Approach 1:
The patent uses a linker molecule as an intermediary to connect the gRNA and NLS peptide. This linker facilitates the conjugation process by providing a standardized interface for chemical attachment, thereby simplifying the manufacturing process compared to direct conjugation methods. The linker acts as a mediator that enables reliable conjugation while maintaining the functional properties of both components.
Data Source
AI summary
The present invention provides, among other things, a guide RNA conjugated to a NLS sequence (NLS-gRNA) and method for making and using the same. For example, in some embodiments, the 3′ end of the gRNA is conjugated to the N-terminus of a nuclear localization sequence (NLS) via a linker comprising a chemical moiety and a peptide spacer.