Bioorthogonal sgRNA Suppression for Temporal CRISPR-Cas9 Control
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Solution Overview
Problem
The challenge in CRISPR-Cas9 technology is the persistence of sgRNA-Cas9 ribonuclear proteins in the body, leading to off-target editing at sites with mismatches, necessitating temporal control of CRISPR-Cas9 nuclease activity to prevent further unintended editing.
Innovation Solution
A method involving bioorthogonal chemistry is used to covalently tag single guide RNA (sgRNA) with a reactive group, followed by a small molecule suppressor with a complementary reactive group to inhibit CRISPR-Cas9 nuclease activity through a bioorthogonal click reaction, allowing precise temporal control of genome editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas9 ribonuclear protein is administered to edit target genes, then genome editing efficiency is improved, but off-target editing occurs at unintended sites
Solution Approach 1:
The patent applies preliminary action by pre-tagging the sgRNA with a bioorthogonal reactive group (such as azide or alkylne) before administering the CRISPR-Cas9 complex to the cell. This preliminary modification enables subsequent temporal control through a second bioorthogonal reaction with a suppressor molecule, allowing the editing activity to be controlled and terminated at predetermined time points to prevent off-target effects while maintaining initial editing efficiency.
2Duration of action of moving object
If CRISPR-Cas9 activity is prolonged to ensure complete on-target editing, then editing completeness is improved, but persistence of sgRNA-Cas9 leads to increased off-target effects
Solution Approach 1:
The patent implements periodic action through temporal control of CRISPR-Cas9 activity using a two-stage approach: first, the tagged sgRNA-Cas9 complex performs on-target editing; second, at a predetermined time point, a bioorthogonal suppressor molecule is introduced to terminate activity. This periodic on-off control ensures complete on-target editing while limiting the duration of activity to prevent off-target cleavage events.
Solution Approach 2:
The patent uses a bioorthogonal suppressor molecule as an intermediary to control CRISPR-Cas9 activity. The suppressor contains a reactive group that specifically reacts with the pre-tagged sgRNA, forming a complex that prevents Cas9 from binding to DNA. This intermediary mechanism allows precise temporal termination of activity without directly affecting the Cas9 protein, enabling controlled cessation of editing to eliminate off-target effects.
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
This approach enables reversible, small-molecule-based control of CRISPR-Cas9 activity, minimizing off-target effects by sterically hindering the sgRNA-Cas9 interaction, thus providing precise temporal regulation of gene editing.
Implementation Method 1
The first and second reactive groups are members of a bioorthogonal click chemistry pair that react under physiological conditions to form a covalent adduct, thereby inhibiting CRISPR-Cas9 nuclease activity.
Data Source
AI summary
Disclosed are systems and methods for reversible, chemically inducible regulation of CRISPR-Cas9 activity using bioorthogonal chemistry. The invention includes modified guide RNAs (sgRNAs) incorporating a first bioorthogonal reactive group that does not interfere with native Cas9 function. Upon introduction of a CRISPR suppressor comprising a second complementary bioorthogonal reactive group, a rapid and selective chemical reaction occurs under physiological conditions, resulting in inhibition of Cas9-mediated genome editing. The suppressors may include small molecules, peptides, or nucleic acid analogs designed to sterically or conformationally disrupt the ribonucleoprotein complex. This platform enables precise spatial and temporal control over CRISPR-Cas9 activity, with potential applications in improving editing specificity and reducing off-target effects. The disclosed methods are modular, biocompatible, and adaptable to a variety of gene editing contexts.