Cas9 Nuclease Segmentation for Off-Target Reduction
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Solution Overview
Problem
The CRISPR-Cas9 system faces challenges in reducing off-target nucleic acid double-stranded breaks and enhancing homology-directed repair efficiency, as it can introduce unintended DNA cleavages and has inefficient site-directed genome editing due to non-homologous end joining and off-target effects.
Innovation Solution
A method involving a complex of catalytically active Cas9 and guide RNA targeting a specific nucleic acid, alongside a complex of catalytically inactive dCas9 and guide RNA to prevent off-target cleavage, using mutated Cas9 proteins with reduced nuclease activity to bind and cleave specific strands of DNA, thereby reducing off-target binding and enhancing HDR efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas9 is used to perform site-directed genome editing, then genome engineering efficiency is improved, but off-target nucleic acid double-stranded breaks occur
Solution Approach 1:
The invention divides the Cas9 nuclease into two separate catalytic domains: the HNH domain that cleaves the complementarily paired strand and the RuvC domain that cleaves the non-complementarily paired strand. By separating these functions into distinct domains that can be independently regulated, the system achieves precise control over DNA cleavage, reducing off-target effects while maintaining on-target editing efficiency
Solution Approach 2:
The invention introduces a single-stranded DNA intermediary that serves as a bridge between the HNH and RuvC domains. This intermediary is recruited to the target site by the HNH domain and subsequently cleaved by the RuvC domain, creating a controlled intermediate step that enhances specificity and reduces off-target cleavage events
2Reliability
If non-homologous end joining repairs DNA double-stranded breaks, then DNA repair occurs, but homology-directed repair efficiency is reduced
Solution Approach 1:
The invention creates single-stranded breaks before introducing the homology donor template, preparing the DNA ends in advance for homologous recombination. This preliminary action of creating controlled breaks with specific overhangs facilitates the subsequent binding and integration of the donor template, thereby enhancing homology-directed repair efficiency
Solution Approach 2:
The invention modifies the DNA break parameters by creating single-stranded breaks with specific overhang configurations rather than blunt double-stranded breaks. By changing the break type and geometry, the system favors homology-directed repair over non-homologous end joining, as the overhanging ends are more compatible with homologous recombination mechanisms
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 effectively minimizes off-target genome editing events and increases the efficiency of targeted nucleic acid modifications by reducing unintended cleavages and improving the precision of homology-directed repair.
Implementation Method 1
The RNA-guided Cas9 endonuclease specifically targets and cleaves DNA in a sequence-dependent manner
Implementation Method 2
the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-part RNA structure, also called 'dual-guide,' that directs the CRISPR-associated protein Cas9
Data Source
Figure 1A~1B
Figure 2
Figure 3A
AI summary
Methods for use with Type II CRISPR-Cas9 systems for increasing Cas9-mediated genome engineering efficiency are disclosed. The methods can be used to decrease the number of off-target nucleic acid double-stranded breaks and/or to enhance homology-directed repair of a cleaved target nucleic acid.