Covalently Linked CRISPR Complexes for Stable Gene Editing
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Solution Overview
Problem
The CRISPR/Cas system faces challenges with the instability of CRISPR complexes, leading to degradation and immune responses, which limits their application due to partial or full dissociation in vivo, resulting in reduced efficiency and off-target cleavage events. There is a need for precise delivery of CRISPR Cas enzyme and guide RNA molecules with enhanced stability and tunable activity.
Innovation Solution
A CRISPR complex is developed with a single guide RNA (sgRNA) cross-linked to a CRISPR effector protein at an unnatural nucleotide outside the target binding region, using modifications such as uracil or maleimide, to maintain nuclease activity and reduce off-target effects, and is formulated with a pharmaceutically acceptable excipient for administration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR complexes are delivered as pure reagents with precise ratios, then dosing accuracy is improved, but stability and resistance to degradation worsen
Solution Approach 1:
The patent modifies the chemical parameters of the guide RNA by incorporating unnatural nucleotides with specific chemical groups (maleimide, pyridyl disulfide, NHS ester, etc.) that enable covalent bonding to the Cas enzyme. This chemical modification changes the stability parameters of the CRISPR complex, allowing it to resist degradation while maintaining precise dosing characteristics.
Solution Approach 2:
The patent creates a composite structure by covalently linking the guide RNA to the Cas enzyme through crosslinking chemistry. This forms a stable复合 material where the RNA-protein complex is chemically bonded, preventing dissociation and degradation while maintaining the functional precision of the CRISPR system.
2Productivity
If CRISPR complexes remain stable in vivo, then efficiency is improved, but off-target cleavage events increase due to dissociation when unstable
Solution Approach 1:
By modifying the chemical composition of the guide RNA with unnatural nucleotides and changing the bonding parameters from non-covalent to covalent interactions, the patent stabilizes the CRISPR complex in vivo. This prevents premature dissociation that would lead to off-target effects, thereby improving on-target editing efficiency while reducing harmful off-target cleavage events.
Solution Approach 2:
The patent creates a stable, reusable CRISPR complex that maintains its integrity throughout the therapeutic window. The covalently linked complex resists degradation and dissociation, allowing it to function reliably as a disposable therapeutic agent with consistent performance rather than degrading into harmful off-target components.
3Adaptability or versatility
If synthetic guide RNAs are used to recruit Cas enzyme to different target sites, then versatility is improved, but immune response increases limiting application
Solution Approach 1:
The patent changes the chemical parameters of the guide RNA by incorporating modified nucleotides and unnatural bases. These chemical modifications alter the immunogenicity parameters of the RNA while preserving its ability to recruit Cas enzymes to different target sites, thereby reducing immune responses without sacrificing versatility.
4Stability of the object's composition
If CRISPR complexes are delivered encoded in plasmids relying on transcription, then stability is improved, but dosing precision worsens due to variable expression
Solution Approach 1:
The patent performs preliminary action by pre-assembling the CRISPR complex with covalently linked guide RNA and Cas enzyme before delivery. This pre-formulation ensures precise dosing ratios are established upfront, and the covalent bonding maintains this precision throughout delivery and cellular uptake, avoiding the variable expression problems of plasmid-based systems.
Solution Approach 2:
The patent creates a stable composite material where the guide RNA and Cas enzyme are covalently bonded. This composite structure maintains its integrity during delivery, ensuring that the precise stoichiometric ratios established during manufacturing are preserved in vivo, unlike plasmid systems where expression ratios can vary.
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 cross-linked CRISPR complex achieves enhanced stability and reduced off-target activity, allowing for precise editing of nucleic acid molecules with high viability of edited cells, maintaining nuclease activity, and improved dosing accuracy.
Implementation Method 1
a single guide RNA (sgRNA) cross-linked to a CRISPR effector protein at an unnatural nucleotide within the sgRNA
Implementation Method 2
The unnatural nucleotide can comprise a maleimide. The maleimide can covalently link to a cysteine on 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.