Artificial Nucleic Acid Inhibitors for CRISPR Safety
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Solution Overview
Problem
Current CRISPR technologies face challenges with off-target effects and safety concerns, necessitating the development of effective inhibitors to control CRISPR enzyme activity and prevent unforeseen adverse events.
Innovation Solution
Development of artificial nucleic acid constructs that interact with specific domains of CRISPR-associated proteins and guide RNAs, including the protospacer adjacent motif (PAM) domain, guide sequence, and tracrRNA repeat region, to inhibit CRISPR enzyme activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If CRISPR enzymes are used for gene editing, then therapeutic potential and ability to cure genetic disease are improved, but off-target effects and safety concerns worsen
Solution Approach 1:
The patent introduces anti-CRISPR proteins as intermediary molecules that specifically bind to and inhibit CRISPR-Cas9 enzyme activity. These proteins act as mediators between the CRISPR system and the cellular environment, providing controlled suppression of off-target effects while allowing on-target editing to proceed when needed
Solution Approach 2:
The patent employs small molecule compounds that modify the functional parameters of CRISPR enzymes through direct binding. These compounds change the enzymatic activity parameters (such as catalytic rate and substrate binding affinity) to reduce off-target effects while maintaining sufficient on-target activity
2Object-generated harmful factors
If natural anti-CRISPR proteins are used to inhibit CRISPR, then off-target effects are reduced, but molecule size and delivery complexity increase
Solution Approach 1:
The patent extracts and utilizes the essential inhibitory function of natural anti-CRISPR proteins by identifying and applying small molecule compounds that replicate this inhibition capability. This extraction approach isolates the key functional property (CRISPR inhibition) from the complex protein structure, enabling simpler molecular designs
Solution Approach 2:
The patent creates simplified copies of the anti-CRISPR function using small molecule compounds that mimic the inhibitory behavior of natural anti-CRISPR proteins. These molecular copies replicate the essential binding and inhibition properties without requiring the full protein structure, thereby reducing molecular complexity
Data Source
AI summary
A CRISPR inhibitor molecule is provided, comprising an artificial nucleic acid construct having a first polynucleotide, the inhibitor molecule capable of establishing several points of contact with a CRISPR protein and high binding affinity thereto, is provided. The first polynucleotide may comprise a sequence selected from the group consisting of: a polynucleotide that interacts with a protospacer adjacent motif (PAM)-interaction (PI) domain of a CRISPR-associated (Cas) protein, a polynucleotide that interacts with a guide sequence of a crRNA or an equivalent position of a single-guide RNA, and a polynucleotide that interacts with a repeat region of a tracrRNA or an equivalent position of a single-guide RNA. The CRISPR inhibitor molecule may also comprise a second polynucleotide and a linker. Methods of using the CRISPR inhibitor molecule in therapeutic agent selection and creation, as well as part of a therapeutic treatment, are also provided.