CRISPR Screening System for On-Target and Off-Target Activity
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Solution Overview
Problem
The CRISPR-Cas system often causes off-target effects, leading to unintended modifications of genes or nucleic acids, which can result in genetic defects and reduce the accuracy and specificity of gene editing processes.
Innovation Solution
A screening system is developed to select target-specific nucleases with high specificity and activity using a multiple target system that simultaneously identifies on-target and off-target activities, incorporating a CRISPR-Cas system with guide RNAs and CRISPR enzymes, and includes selection elements to differentiate between on-target and off-target effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the CRISPR-Cas system is used to modify genes or nucleic acids, then the efficiency of gene modification is improved, but off-target effects occur causing unintended modifications of non-targeted genes
Solution Approach 1:
The screening system segments the evaluation process into distinct on-target and off-target assessment components. By using multiple target sites (including both on-target and off-target sequences) in a single screening assay, the system can simultaneously evaluate both the desired gene modification efficiency and the unwanted off-target effects, allowing selection of nucleases that optimize the ratio between on-target activity and off-target activity.
Solution Approach 2:
The invention employs parameter changes by varying nuclease concentration, incubation time, and target sequence composition in the screening system. By systematically adjusting these parameters and measuring their effects on both on-target and off-target activities, the system identifies optimal conditions that maximize gene modification efficiency while minimizing off-target effects.
2Productivity
If the concentration of Cas9 is increased to improve on-target effect, then gene modification efficiency is improved, but off-target effects are also increased
Solution Approach 1:
The screening system provides feedback by simultaneously measuring both on-target and off-target activities at various Cas9 concentrations. This dual-measurement feedback allows identification of the optimal Cas9 concentration range where on-target effect is maximized while off-target effect remains below acceptable thresholds, enabling precise control of the gene editing process.
Solution Approach 2:
The invention applies partial action by using truncated guide RNAs (reduced length compared to standard guide RNAs) to achieve sufficient on-target binding while reducing off-target interactions. This partial modification of the guide RNA structure allows the system to maintain effective gene editing at lower Cas9 concentrations, thereby reducing off-target effects.
3Manufacturing precision
If guide RNA sequences are modified to reduce off-target effects, then specificity is improved, but on-target activity may be reduced
Solution Approach 1:
The screening system evaluates multiple guide RNA variants with different sequences, lengths, and structural modifications. By systematically changing guide RNA parameters and measuring their impact on both on-target activity and off-target specificity using the multiple target system, the invention identifies optimal guide RNA designs that achieve high specificity without sacrificing on-target activity.
Solution Approach 2:
The invention applies local quality by making specific modifications to particular regions of the guide RNA (such as the seed region or proximal/distal regions) rather than uniformly altering the entire sequence. This localized modification approach allows optimization of specific functional aspects (binding affinity, specificity) while preserving other important functions, achieving high on-target activity with reduced off-target effects.
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
The system effectively reduces off-target effects and increases on-target specificity, allowing for precise gene editing by identifying and selecting nucleases with minimized off-target activity and maximized on-target activity.
Implementation Method 1
a guide RNA having a sequence complementary to a gene or nucleic acid to be targeted and a CRISPR enzyme which is an enzyme cleaving the gene or nucleic acid to be targeted, and the guide RNA and the CRISPR enzyme form a CRISPR complex
Implementation Method 2
the CRISPR enzyme which is an enzyme cleaving the gene or nucleic acid to be targeted
Implementation Method 3
The gene or nucleic acid that is not targeted can partially interact with the guide RNA by including a sequence partially complementary to the guide RNA
Data Source
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AI summary
The present invention relates to a method for selecting targeted genetic scissors having high specificity and high activity and, more particularly, to a method for screening and selecting a targeted genetic scissors system by using a multiple target system capable of simultaneously identifying off-target activity and on-target activity. Through the present invention, selection can be made of targeted genetic scissors which are decreased in off-target effect and increased in on-target effect.