Anti-CRISPR Proteins for Cas9 Activity Downmodulation
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Solution Overview
Problem
There is a need to discover additional anti-CRISPR proteins and genes with a broad spectrum of action against various bacterial species and highly effective against CRISPR-Cas systems to modulate their activity and provide resistance against virulent phages.
Innovation Solution
The development of anti-CRISPR proteins and genes that interfere with the function of CRISPR-Cas systems, specifically targeting class 2 type II CRISPR-Cas systems in bacteria, such as Streptococcus thermophilus and Streptococcus pyogenes, by downmodulating the activity of Cas9 proteins and reducing phage resistance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If CRISPR-Cas system activity is enhanced to improve genome-editing efficiency, then off-target effects and immune resistance increase, but precision and safety decrease
Solution Approach 1:
The patent applies preliminary anti-action by introducing anti-CRISPR proteins that preemptively inhibit CRISPR-Cas system activity. These proteins are expressed alongside CRISPR components to prevent excessive or off-target cleavage activity before it occurs, thereby maintaining safety while allowing controlled editing efficiency
Solution Approach 2:
The patent employs parameter changes by modulating CRISPR-Cas system activity through anti-CRISPR proteins that alter the functional state of Cas enzymes. This dynamic regulation allows the system to switch between active and inactive states, enabling precise control over editing timing and location while preventing unwanted off-target effects
2Adaptability or versatility
If broad-spectrum anti-CRISPR proteins are developed to increase resistance against multiple bacterial species, then the spectrum of protection expands, but specificity and targeted effectiveness may decrease
Solution Approach 1:
The patent applies universality by developing anti-CRISPR proteins that can inhibit multiple CRISPR-Cas system variants across different bacterial species. These proteins are designed with broad binding specificity that allows them to recognize and inhibit conserved structural features of Cas enzymes from type II-A, type II-B, and type II-C systems, providing multi-functional protection
Solution Approach 2:
The patent employs segmentation by identifying and targeting specific functional domains within CRISPR-Cas systems that are conserved across species. By focusing on essential catalytic domains or RNA-binding interfaces that are common to multiple system types, the anti-CRISPR proteins achieve broad spectrum inhibition while maintaining reasonable specificity for CRISPR-related functions
3Productivity
If anti-CRISPR proteins are used to downmodulate CRISPR-Cas activity to increase phage titer, then phage propagation improves, but bacterial immune resistance decreases
Solution Approach 1:
The patent applies periodic action by using anti-CRISPR proteins that are expressed in a temporally controlled manner. The proteins are activated at specific stages of the phage lifecycle to allow phage propagation during permissive phases, while CRISPR immunity is restored during restrictive phases, creating a periodic cycle of susceptibility and resistance that enables controlled phage production
Data Source
Figure 1
Figure 2A~2D
Figure 3A~3B
AI summary
The invention relates to anti-CRISPR genes and anti-CRISPR proteins, and their uses in various biotechnology applications.