Anti-CRISPR Agents Control CRISPR Activity Timing in Human Embryos
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Solution Overview
Problem
Current CRISPR-Cas9 gene editing in human embryos faces challenges with mosaicism and off-target effects due to the timing of Cas9 activity, which complicates the precise editing needed for heritable genetic diseases.
Innovation Solution
The use of anti-CRISPR agents, specifically proteins like AcrIIA4, is introduced after gene editing to control and inhibit CRISPR activity, reducing mosaicism and off-target effects by timing their administration to coincide with the first cell cycle or shortly after Cas9-mediated cleavage.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR-Cas9 activity is maintained for extended periods to ensure complete gene editing, then editing completeness is improved, but mosaicism and off-target effects increase
Solution Approach 1:
The anti-CRISPR agent is introduced in advance or simultaneously with CRISPR-Cas9 components, establishing a controlled inhibition mechanism that prevents prolonged Cas9 activity. This preliminary action ensures that Cas9 is rapidly turned off after initiating editing, reducing mosaicism and off-target effects while maintaining editing completeness through the initial cleavage event.
Solution Approach 2:
The anti-CRISPR agent serves as an intermediary that mediates between CRISPR-Cas9 editing activity and cellular processes. By introducing this intermediary inhibitor, the system achieves precise temporal control where Cas9 activity is activated for the necessary editing window and then rapidly terminated, resolving the contradiction between editing completeness and harmful effects.
2Loss of time
If CRISPR-Cas9 activity occurs after the first S-phase, then more time is available for editing, but the likelihood of mosaicism increases due to multiple chromosome copies
Solution Approach 1:
The system performs the gene editing action preliminarily during the zygotic stage before the first S-phase occurs. By introducing CRISPR-Cas9 components at fertilization and using anti-CRISPR to limit activity duration, the editing is completed while there is still a single copy of each chromosome, ensuring uniform editing across all future cells and eliminating mosaicism.
3Object-affected harmful factors
If anti-CRISPR agents are introduced too early, then Cas9 activity is suppressed, but on-target editing efficiency decreases
Solution Approach 1:
The system employs dynamic temporal control where anti-CRISPR agent timing is optimized to achieve the right balance. The agent is introduced at a specific time point that allows sufficient Cas9 activity for complete on-target editing while subsequently suppressing further activity to prevent off-target effects. This dynamic approach adjusts the inhibition timing to match the editing kinetics.
Solution Approach 2:
The system uses periodic or pulsed CRISPR-Cas9 activity controlled by anti-CRISPR timing. Cas9 is activated for a brief initial period to perform on-target editing, then anti-CRISPR is introduced to terminate activity. This periodic action pattern ensures complete editing during the active phase while minimizing harmful effects during the inhibited phase.
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 significantly reduces on-target and off-target indels by approximately 45% and 74-79%, respectively, and minimizes mosaicism, enhancing the efficiency and specificity of gene editing in human embryos.
Implementation Method 1
anti-CRISPR proteins, were first discovered in viruses in 2013... Shin et al. 2017 showed that the anti-CRISPR protein, AcrIIA4 was able to bind and inhibit CRISPR-Cas9 activity in human cells
Data Source
AI summary
The present disclosure relates to using anti-CRISPR agents and methods to control, reduce and/or inhibit gene editing, thus reducing, eliminating and/or preventing mosaicism and off-target effects of gene editing in embryos. The gene editing can be performed using CRISPR technology and the CRISPR technology can be a CRISPR/Cas9 system.