Nucleic Acid Editing Off-Target Detection via Statistical Modeling
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Solution Overview
Problem
Current nucleic acid editing techniques, such as CRISPR-based genome editing systems, lack specificity and accuracy, leading to off-target activity and unwanted genome editing events, which can result in genomic instability and adverse effects like mutations and translocations, limiting their safe use in clinical and agricultural applications.
Innovation Solution
A novel technique using the treatment vs. mock approach with advanced modeling of multiplex-amplification PCR data to accurately detect and quantify off-target activities, including indels and translocations, by analyzing sequencing data from edited and control samples to determine the actual occurrence of adverse effects.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If nucleic acid editing techniques are used to achieve genome editing, then genetic illnesses can be cured and genome editing can be achieved, but off-target activity occurs leading to unwanted genome editing events and genomic instability
Solution Approach 1:
The patent applies preliminary action by performing whole-genome sequencing of the target organism before and after CRISPR-Cas9 editing. This pre-sequencing establishes a baseline genome sequence, allowing researchers to identify and compare potential off-target mutations that occur during editing. The pre-prepared reference genome enables systematic detection of unintended genetic changes by comparing post-editing sequences against the established baseline.
Solution Approach 2:
The patent implements feedback through comprehensive sequencing analysis that identifies off-target mutations and provides this information back to the editing process. By sequencing the entire genome and comparing it to the reference sequence, the system generates feedback data about unintended edits. This feedback mechanism allows researchers to assess the specificity of CRISPR-Cas9 editing and make adjustments to guide RNA design or editing parameters to reduce off-target effects in subsequent experiments.
2Measurement precision
If comprehensive sequencing analysis is performed to detect off-target effects, then accuracy of editing assessment is improved, but time and resources required for analysis increase
Solution Approach 1:
The patent applies segmentation by dividing the genome into manageable sequencing segments and analyzing them systematically. Rather than attempting to analyze the entire genome as a single unit, the sequencing process is broken down into smaller, parallelizable segments that can be processed independently. This segmentation allows for more efficient use of sequencing resources and enables parallel processing, reducing the overall time required while maintaining comprehensive coverage for detecting off-target effects.
Data Source
AI summary
Novel systems methods and kits are disclosed for determining effects of Nucleic Acid (NA) editing procedures. The technique (methods systems and kits) of the present invention are adapted for receiving sequencing data indicative of pluralities of reads, RTx={riTx} and RMc={rjMc}, of the multiplexed amplifications of each of the edit and control NA collections, originating from a similar source of NAs whereby a certain NA editing procedure was applied to the edit NA collection; and processing the sequencing data, per each particular type of adverse effect of interest to determine a statistical model for classifying whether that type of adverse effect actually occurred due to the NA editing procedure. The technique is suitable for determining occurrences of INDEL types and/or TRANSLOCATION types/species of adverse effect. The technique further enables to statistically quantify the rates of occurrence types of adverse effects which are actually caused by the NA editing procedure, and also to provide statistical confidence intervals for these rates according to a desired statistical confidence level.