PASEA Enrichment of Rare Mutant Alleles
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Solution Overview
Problem
Current methods for detecting rare somatic mutant alleles in cancer diagnosis face challenges due to their low abundance and sequence homology with wild-type nucleic acids, requiring laborious, time-consuming, and expensive deep next-generation sequencing for high sensitivity.
Innovation Solution
The Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) method, which uses CRISPR-Cas9 to selectively cleave wild-type alleles, combined with isothermal recombinase polymerase amplification, preferentially amplifies mutant alleles, increasing their frequency to detectable levels using inexpensive sequencers like Sanger in a single step.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If deep next-generation sequencing is used to detect rare mutant alleles, then detection sensitivity is improved, but the method becomes laborious, time-consuming, and expensive
Solution Approach 1:
The detection process is segmented into two distinct stages: (1) CRISPR-Cas9-mediated selective depletion of wild-type nucleic acids, and (2) exponential amplification of the remaining mutant alleles. This segmentation allows each stage to optimize for its specific function, achieving high sensitivity without requiring laborious deep sequencing
Solution Approach 2:
The CRISPR-Cas9 system performs preliminary selective depletion of abundant wild-type nucleic acids before amplification. By removing the background wild-type sequences in advance, the subsequent amplification step can focus exclusively on enriching mutant alleles, dramatically improving detection efficiency and reducing costs
2Measurement precision
If CRISPR-Cas9 is used to remove background nucleic acids and enrich rare mutant allele fractions, then detection sensitivity is improved, but the method is limited by absence of restriction endonuclease recognition sites, futile binding events, and off-target cleavage
Solution Approach 1:
The invention merges CRISPR-Cas9 selective depletion with exponential amplification into a unified two-stage workflow. The amplification step compensates for incomplete depletion efficiency and mitigates the impact of off-target cleavage by exponentially enriching mutant alleles, thereby maintaining high assay accuracy despite limitations of CRISPR-Cas9 alone
Solution Approach 2:
The invention changes the operational parameters by introducing exponential amplification conditions (polymerase, nucleotides, thermal cycling) after CRISPR depletion. This parameter change transforms the system from simple depletion to selective enrichment, overcoming limitations of recognition site availability and binding efficiency
3Measurement precision
If rounds of selective depletion of wild-type followed with PCR are performed, then some shortcomings are overcome, but high sensitivity detection still requires deep NGS rendering methods laborious, time-consuming, and expensive
Solution Approach 1:
The invention maintains continuous useful action by immediately following CRISPR-mediated depletion with exponential amplification of mutant alleles. This continuous workflow eliminates idle time between depletion and detection, and the exponential nature of amplification rapidly increases mutant allele frequency to detectable levels within hours rather than requiring time-consuming deep sequencing
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
PASEA achieves high sensitivity and specificity in detecting mutant alleles, enabling the enrichment of rare somatic mutant alleles to nearly 100% of amplicons, allowing for real-time detection and reducing the need for deep sequencing, making it suitable for point-of-care applications and resource-limited settings.
Implementation Method 1
an endonuclease having an affinity for the guide/non-target hybrid
Implementation Method 2
amplifying the target nucleic acid in an amplification reaction comprising a polymerase
Data Source
AI summary
Described is an assay termed Programmable Enzyme-Assisted Selective Exponential Amplification (PASEA) that concurrently amplifies both wild type and mutant alleles while selectively cleaving the former. With time, the rare mutant alleles dominate, and are readily detectable by direct detection, Sanger sequencing, and other readily available methods. Also described are point-of-care assays and microfluidic devices for performing PASEA.