CRISPR Genomic Excision for Complex Locus Sequencing
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Solution Overview
Problem
Current methods for analyzing complex genomic regions, such as the CYP2D6 locus, are prone to mischaracterization due to the complexity of genetic variations and the limitations of existing genotyping platforms, leading to incorrect dosing recommendations and increased risk of adverse drug reactions.
Innovation Solution
A method involving the use of a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and guide RNAs to excise and analyze genomic regions of interest without DNA amplification, utilizing long-read sequencing for accurate genotyping and structural analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current commercial genotyping platforms are used to analyze CYP2D6, then the testing process is simple and fast, but the accuracy is poor leading to mischaracterization of genetic variants
Solution Approach 1:
The method segments the complex CYP2D6 genomic region into manageable components by using long-read sequencing to capture and analyze the entire locus including pseudogenes as a single unit, rather than attempting to genotype individual variants separately. This segmentation approach allows accurate characterization of complex structural variants while maintaining workflow feasibility.
Solution Approach 2:
The patent introduces long-read sequencing technology as an intermediary between sample collection and variant calling. This intermediary technology bridges the gap by providing sufficient read lengths to span the entire CYP2D6 locus and its pseudogenes, enabling accurate detection of structural variants without requiring complex multi-step genotyping workflows.
2Measurement precision
If gene sequencing based on short reads (NGS) or template length (Sanger sequencing) is used, then the equipment and reagents are readily available, but the ability to accurately analyze complex haplotypes is limited
Solution Approach 1:
The method changes the critical parameter of read length from short (hundreds of bases in NGS) or limited template length (Sanger sequencing) to long reads that can span the entire CYP2D6 locus. This parameter change enables accurate resolution of complex haplotypes and structural variants while the patent addresses equipment availability through targeted approaches that can be implemented on emerging long-read platforms.
3Measurement precision
If methods combining targeted amplification, copy number analysis, and long-range PCR are used, then the accuracy of determining full structure is improved, but the workflow complexity, time requirements, and cost increase making them unsuitable for routine clinical testing
Solution Approach 1:
The patent merges multiple separate analytical steps (targeted amplification, copy number analysis, long-range PCR) into a single unified long-read sequencing approach. By combining these functions into one technology platform, the method maintains high accuracy for structural variant determination while dramatically simplifying the workflow and enabling routine clinical throughput.
Solution Approach 2:
The method extracts and analyzes the entire CYP2D6 locus including pseudogenes as a single continuous DNA molecule through long-read sequencing. This extraction approach eliminates the need for multiple separate amplification and analysis steps, reducing workflow complexity while maintaining comprehensive structural analysis capability.
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 improves the accuracy and reduces the time and cost of sequencing complex genomic regions, providing precise genetic information for personalized medicine and minimizing the risk of adverse drug reactions.
Implementation Method 1
contacting genomic DNA comprising the genomic region of interest with a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and two or more gRNAs, thereby generating an excised genomic region of interest
Data Source
AI summary
Provided herein are methods of genotyping complex genomic regions. In some cases, the methods involve the use of a CRISPR-associated endonuclease and two or more guide RNAs to excise a genomic region of interest from genomic DNA. The methods further involve the use of long-read sequencing to sequence the genetic region of interest. In some cases, the methods are amplification-free.