Covalent CRISPR-Cas Complexes for Stable Guide RNA Delivery
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current CRISPR/Cas systems face issues with instability and off-target cleavage due to degradation of synthetic guide RNAs and immune responses, necessitating precise delivery and enhanced stability for effective gene editing.
Innovation Solution
A CRISPR complex is developed with a single guide RNA (sgRNA) conjugated to a CRISPR effector protein at an unnatural nucleotide, using modifications like uracil or thymidine with a maleimide linkage to enhance stability and reduce off-target nuclease activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If synthetic guide RNA is used to form CRISPR complexes, then the system can be delivered as a pure reagent with precise dosing, but the guide RNA is subject to degradation when not in complex with Cas enzyme, reducing stability
Solution Approach 1:
The patent merges the guide RNA and Cas enzyme into a single conjugated complex through chemical linkage. The guide RNA is covalently attached to the Cas enzyme at specific cysteine residues, creating a stable pre-formed complex that maintains both delivery precision and molecular stability. This eliminates the need to deliver separate components that might degrade or dissociate.
Solution Approach 2:
The CRISPR complex is pre-assembled and conjugated before delivery to the target cell. The guide RNA is chemically modified and linked to the Cas enzyme in advance, creating a stable pre-formed complex. This preliminary action ensures the complex remains intact during delivery and can immediately function upon entering the target cell, avoiding degradation that would occur with separate components.
2Reliability
If synthetic guide RNA is used to form CRISPR complexes, then the system can be delivered with precise ratios, but the guide RNA can induce an immune response which limits application
Solution Approach 1:
The patent modifies the chemical parameters of the guide RNA by incorporating unnatural nucleotides and chemical modifications. These parameter changes alter the immunogenicity of the guide RNA, reducing its ability to trigger immune responses while maintaining its guide function. The modified nucleotides change the molecular recognition properties that immune systems typically detect.
Solution Approach 2:
The patent converts the potential harm of guide RNA immunogenicity into a benefit by using the same chemical modification approach to improve stability and reduce immune recognition. The modifications that were initially designed to improve conjugation efficiency also inadvertently reduce immunogenicity, turning a harmful property into a beneficial one.
3Ease of manufacture
If CRISPR complexes are delivered as separate components requiring transcription, then plasmid delivery can be used, but the complexes can dissociate in vivo reducing efficiency and causing off-target cleavage
Solution Approach 1:
The patent merges the guide RNA and Cas enzyme into a single conjugated complex through chemical linkage. This merging prevents dissociation of the complex components in vivo, maintaining the integrity of the CRISPR system throughout delivery and function. The covalent bond between guide RNA and Cas enzyme eliminates the risk of dissociation that plagues separate component delivery.
Solution Approach 2:
The patent creates a composite CRISPR complex by chemically linking the guide RNA and Cas enzyme into a single hybrid molecule. This composite structure combines the advantages of both components while eliminating their disadvantages - the stability of the protein is enhanced by the RNA linkage, and the RNA guidance function is stabilized by the protein attachment, creating a more reliable complex than either component alone.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The modified CRISPR complex achieves high efficiency and viability in gene editing, with less than 2% off-target cleavage and 99% viable edited cells, maintaining nuclease activity and stability.
Implementation Method 1
The unnatural nucleotide can comprise a maleimide. The maleimide can covalently link to a cysteine on the CRISPR effector protein thereby conjugating the unnatural nucleotide to the CRISPR effector protein.
Data Source
AI summary
Provided herein are polynucleotides and CRISPR effector proteins configured to be covalently bound together in a CRISPR complex. The polynucleotides can be further modified to modulate the activity of the CRISPR complex. Modification of the polynucleotide and CRISPR effector protein can be used to improve the efficacy of target binding and/or cleavage.