Anti-CRISPR Proteins Modulate Cas Endonuclease Specificity
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Solution Overview
Problem
Current genome-editing tools, such as CRISPR systems, face challenges with specificity and efficiency, particularly in plants, leading to off-target effects and the need for costly and time-consuming redesigns for each target site.
Innovation Solution
The use of anti-CRISPR (ACR) proteins to modulate Cas endonuclease activity in plant cells, combined with guide RNAs, to improve specificity and reduce off-target cleavage, allowing for controlled and targeted gene editing by regulating Cas endonuclease activity temporally and spatially.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR systems are used for genome editing in plants, then editing efficiency is improved, but off-target effects increase and specificity decreases
Solution Approach 1:
The patent introduces anti-CRISPR proteins as intermediary molecules that bind to Cas endonucleases and modulate their activity. These ACR proteins act as mediators between the guide RNA-Cas complex and the target DNA, providing an additional layer of control that enhances specificity by preventing off-target binding while allowing on-target editing when properly regulated
Solution Approach 2:
The patent employs parameter changes by using anti-CRISPR proteins to dynamically adjust the activity state of Cas endonucleases. By controlling the concentration, timing, and spatial distribution of ACR proteins, the system can tune Cas enzyme activity levels to achieve optimal balance between editing efficiency and specificity across different cellular conditions
2Productivity
If Cas endonuclease activity is increased to improve editing efficiency, then off-target cleavage increases
Solution Approach 1:
The patent applies preliminary anti-action by introducing anti-CRISPR proteins that preemptively bind to and inhibit Cas endonucleases before they can engage in non-specific DNA binding. This pre-inhibition strategy prevents off-target cleavage events by blocking the Cas enzyme's active site or DNA-binding interface, while allowing controlled activation for on-target editing when needed
3Manufacturing precision
If CRISPR systems are designed for each target site, then specificity is improved, but time and cost increase
Solution Approach 1:
The patent demonstrates universality by using a single Cas endonuclease system (such as Cas9 or Cpf1) that can target multiple different genomic sites through exchange of guide RNA molecules. The addition of anti-CRISPR proteins provides universal control over this system's activity, allowing the same Cas enzyme to be reused across different target sites with appropriate guide RNAs, eliminating the need to redesign the nuclease itself for each target
Data Source
AI summary
Methods and compositions are provided for the use of anti-CRISPR (ACR) proteins in plants, including modulation of Cas endonuclease activity, improvement of frequency of homologous recombination, control of Cas endonuclease activity during various cell cycles, spatial and/or temporal regulation of Cas endonuclease activity in plants, usage in gene activation or repression, as well as reduction of off-target polynucleotide cleavage.