CRISPR-Cas Enrichment for Rare Mutation Detection
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Solution Overview
Problem
Current DNA sequencing methods face challenges in detecting rare mutations due to the stochastic nature of PCR amplification and the abundance of wild-type DNA, often missing mutant alleles present in low frequencies.
Innovation Solution
The use of RNA-guided binding proteins like Cas endonuclease, which protect mutation-containing nucleic acid while digesting non-target DNA, allowing for enrichment and detection of rare mutations even at frequencies as low as 0.01% through specific binding and exonuclease digestion, followed by amplification using phosphorothioate primers to preserve target fragments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If PCR amplification is used to detect rare mutations, then the mutant alleles can be amplified, but the stochastic nature of PCR causes loss of rare fragments
Solution Approach 1:
The patent applies preliminary action by using CRISPR/Cas binding proteins to capture and protect rare mutant DNA fragments before PCR amplification. The binding proteins specifically bind to mutant alleles with high affinity, enriching them in the sample before the amplification step, which prevents the stochastic loss of rare fragments during PCR.
Solution Approach 2:
The patent uses CRISPR/Cas binding proteins as intermediaries between the rare mutant DNA and the detection system. These proteins act as mediators that specifically recognize and bind to mutant alleles, enabling their selective enrichment and subsequent detection while avoiding the stochastic effects of direct PCR amplification of rare targets.
2Measurement precision
If fluorescent probe hybridization is used to detect mutations, then the assay can identify mutant sequences, but probe assays miss mutants present in quantities as low as hundredths of a percent
Solution Approach 1:
The patent applies preliminary action by performing CRISPR/Cas-mediated enrichment of mutant DNA fragments before the detection step. This preliminary enrichment concentrates rare mutants (even at hundredths of a percent frequency) to detectable levels, enabling subsequent precise detection by sequencing or other methods.
Solution Approach 2:
The patent changes the concentration parameter of mutant DNA in the sample through CRISPR/Cas enrichment. By selectively binding and protecting mutant fragments from degradation, the method increases the effective concentration of rare mutants from hundredths of a percent to levels suitable for accurate detection and sequencing.
3Loss of information
If NGS platforms are used to sequence DNA, then comprehensive genomic information can be obtained, but rare mutations are lost among abundant wild-type DNA
Solution Approach 1:
The patent applies the extraction principle by using CRISPR/Cas binding proteins to specifically extract and isolate rare mutant DNA fragments from the abundant wild-type DNA background. The binding proteins selectively bind to mutant alleles, allowing their physical separation and enrichment before NGS, so that rare mutations are no longer lost among overwhelming amounts of wild-type sequences.
4Quantity of substance
If exonuclease digestion is used to remove non-target DNA, then background DNA is reduced, but target fragments may also be degraded without protection
Solution Approach 1:
The patent applies preliminary anti-action by using CRISPR/Cas binding proteins to protect target mutant DNA fragments before exonuclease digestion. The binding proteins bind specifically to mutant alleles and prevent exonuclease access, creating a protective barrier that allows selective removal of non-target DNA while preserving the rare mutant targets.
Solution Approach 2:
The CRISPR/Cas binding proteins serve as intermediaries between the target mutant DNA and the exonuclease. They bind to the target sequences and physically block the exonuclease from degrading the protected fragments, while allowing the exonuclease to freely digest unprotected non-target DNA in the sample.
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 method effectively captures and detects rare mutations in clinical samples, such as those from tumors, by enriching for target fragments and enabling their sequencing or analysis even in samples where mutants are present in very small quantities.
Implementation Method 1
binding a protein to the target nucleic acid in a sequence-specific manner
Implementation Method 2
RNA-guided binding proteins, such as a Cas endonuclease, can bind to, and protect, mutation-containing nucleic acid
Implementation Method 3
An exonuclease is introduced that digests DNA. However, those amplicons that include the mutation and are bound by the Cas complex will not be digested by the exonuclease
Implementation Method 4
amplifying the target nucleic acid with at least one primer that is resistant to degradation by a nuclease to yield an amplicon that includes a copy of the target nucleic acid and a terminal portion that is resistant to degradation by the nuclease
Data Source
AI summary
Methods for detecting rare mutations in DNA include obtaining a sample comprising a target nucleic acid, binding a protein to the target nucleic acid in a sequence-specific manner, digesting non-target nucleic acid in the sample, and detecting the target nucleic acid. The method may include amplifying the target nucleic acid with at least one primer with, e.g., a phosphorothioate bond that is resistant to degradation by a nuclease to yield an amplicon that includes a copy of the target nucleic acid and a terminal portion that is resistant to degradation by the nuclease. Preferably digesting the non-target nucleic acid includes exposing amplicons to the nuclease. The nuclease digests the non-target nucleic acid while the amplicon that includes the copy of the target nucleic acid is protected by the terminal portions and the bound protein.