Blocking Guide RNA for CRISPR Off-Target Suppression
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Solution Overview
Problem
The CRISPR-Cas9 genome editing system suffers from off-target DNA cleavage, leading to unintended mutations, which is a significant concern for applications seeking precise genetic modifications, as existing strategies to improve specificity often compromise on-target cleavage activity.
Innovation Solution
The use of engineered guide RNAs, including targeting and blocking guide sequences, to enhance the specificity of CRISPR-Cas9 systems by preventing off-target nucleic acid cleavage without significantly reducing on-target activity, through the design of guide RNAs that bind to off-target sequences but do not cleave them.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If Cas9 is used to cleave target DNA sequences, then genome editing efficiency is improved, but off-target cleavage occurs leading to unintended mutations
Solution Approach 1:
The patent introduces a blocking guide RNA as an intermediary molecule that binds to off-target DNA sequences and prevents the Cas9-gRNA complex from cleaving them. This mediator selectively inhibits off-target activity while allowing on-target cleavage to proceed, thus resolving the contradiction between editing efficiency and off-target effects
Solution Approach 2:
The blocking guide RNA performs preliminary anti-action by pre-binding to off-target sequences before the Cas9 complex can cleave them. This preventive mechanism blocks harmful off-target cleavage events before they occur, while maintaining the ability of Cas9 to cleave on-target sequences
2Manufacturing precision
If strategies are employed to improve specificity of CRISPR-Cas9, then off-target cleavage is reduced, but on-target cleavage activity is diminished
Solution Approach 1:
The patent segments the guide RNA function into two distinct components: a targeting gRNA for on-target cleavage and a blocking gRNA for off-target protection. This segmentation allows each component to specialize in its function, with the blocking gRNA specifically designed to bind off-target sequences without compromising the targeting gRNA's cleavage activity
Solution Approach 2:
The blocking guide RNA is designed with specific parameter changes including shorter length (10-18 nucleotides) and modified sequence composition compared to standard guide RNAs. These parameter changes enable selective binding to off-target sequences with lower affinity, preventing spurious cleavage while maintaining high-fidelity on-target activity
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 off-target effects while maintaining or improving on-target activity, offering a more precise and efficient genome editing process.
Implementation Method 1
The guide RNA-Cas9 complex identifies and base pairs with its cognate DNA target sequence
Implementation Method 2
Cas9, an RNA-guided DNA endonuclease, to induce a specific double-stranded break (DSB) at DNA target sites
Data Source
AI summary
This invention relates to reagents and methods for increasing specificity and efficiency of genome editing by CRISPR associated (Cas) protein systems, more particularly by Cas:guide RNA complexes, by blocking off-target nucleic acids from cleavage by Cas:guide RNA complexes.