Small-Molecule Inhibitors for RNA-Guided Nucleases
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Solution Overview
Problem
Current CRISPR-Cas systems face challenges with off-target gene editing effects, lack of dose and temporal control, and toxicity issues, particularly for in vivo applications, and existing small-molecule inhibitors are ineffective in controlling wild-type Cas9/Cpf1 variants and are toxic at the organismal level.
Innovation Solution
Development of specific compounds that inhibit RNA-guided endonucleases such as Cas9 and Cpf1 by contacting them with specific chemical inhibitors, which can reversibly modulate their activity, allowing for dosage and temporal control, and are effective across various variants including wild-type and engineered versions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas systems are used for gene editing, then editing capability is improved, but off-target effects increase
Solution Approach 1:
The patent introduces small molecule compounds as intermediary substances that bind to the PAM-interacting domain of Cas9, acting as a mediator to block the interaction between Cas9 and target DNA. This intermediary mechanism prevents off-target binding while preserving on-target editing capability, directly resolving the contradiction between editing effectiveness and off-target effects
Solution Approach 2:
The patent modifies the binding parameters of Cas9 by introducing small molecule inhibitors that alter the affinity and specificity of Cas9 for target DNA. By changing the binding parameters through chemical inhibition, the system achieves higher specificity for on-target sites while reducing off-target binding, thus resolving the contradiction
2Productivity
If CRISPR-Cas systems are used for gene drives, then gene replacement efficiency is improved, but dose and temporal control is lost
Solution Approach 1:
The patent introduces dynamic control to the CRISPR-Cas system by using small molecule inhibitors that can be added or removed at will. This allows the system to switch between active and inhibited states, enabling temporal control of gene drive activity while maintaining high replacement efficiency when activated, thus resolving the contradiction between efficiency and controllability
Solution Approach 2:
The patent enables dose control by varying the concentration of small molecule inhibitors. By adjusting the inhibitor dosage, the system can precisely control the level of Cas9 activity, allowing for fine-tuned gene replacement efficiency while maintaining temporal control through dosage adjustment
3Reliability
If existing small-molecule inhibitors are used, then Cas9 activity is inhibited, but toxicity at organismal level occurs
Solution Approach 1:
The patent optimizes the chemical parameters of small molecule inhibitors to achieve selective binding to the PAM-interacting domain of Cas9 without affecting other cellular processes. By carefully adjusting molecular parameters such as binding affinity and specificity, the inhibitors achieve effective Cas9 inhibition while minimizing off-target toxicity at the organismal level
Data Source
AI summary
The need to control the activity and fidelity of CRISPR-associated nucleases has resulted in the demand for inhibitory anti-CRISPR molecules. Current small-molecule inhibitor discovery platforms are not generalizable to multiple nuclease classes, only target the initial step in the catalytic activity, and require high concentration of nuclease, resulting in inhibitors with suboptimal attributes, including poor potency. Herein, Applicants report a high-throughput discovery pipeline consisting of a FRET-based assay that is generalizable to contemporary and emerging nucleases, operates at low nuclease concentration, and targets all catalytic steps. Applicants applied this pipeline to identify BRD7586, a cell-permeable small-molecule inhibitor of SpCas9, that is 2-fold more potent than current inhibitors. Furthermore, unlike the reported inhibitors, BRD7586 enhanced SpCas9 specificity and its activity was independent of the genomic loci, DNA repair pathway, or mode of nuclease delivery. Overall, these studies describe a general pipeline to identify inhibitors of contemporary and emerging CRISPR-associated nucleases. Described herein are compositions and methods for inhibiting the activity of RNA-guided endonucleases, and methods for identifying such compositions.