rhPCR Primers for Accurate CRISPR Mutation Detection
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Solution Overview
Problem
Current methods for detecting DNA mutations, particularly those introduced by targetable endonucleases like CRISPR Cas9, are either error-prone or require costly and time-consuming next-generation sequencing.
Innovation Solution
The use of high discrimination polymerase mutants and blocked-cleavable RNase H2-dependent PCR (rhPCR) primers that allow mismatches to be placed 5′ or 3′ of the RNA, enabling enhanced mismatch discrimination and reduced primer-dimer formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard allele-specific PCR is used for mutation detection, then the method is simple and widely applicable, but mismatch detection ability is limited due to low discrimination of wild-type DNA polymerases
Solution Approach 1:
The patent introduces RNase H2 as an intermediary enzyme to enhance mismatch detection. The method uses a blocked-cleavable primer with an RNA-DNA hybrid region that is cleaved by RNase H2 only when perfectly matched to the template, thereby indirectly detecting mismatches through selective cleavage rather than direct polymerase discrimination
Solution Approach 2:
The patent changes the chemical composition parameter of the primer by incorporating a blocked-cleavable design with RNA-DNA hybrid structure. This parameter change enables the primer to be selectively cleaved by RNase H2 under specific conditions, transforming the detection mechanism from relying on polymerase discrimination to enzyme-mediated cleavage
2Measurement precision
If the mismatch is placed immediately opposite the RNA in rhPCR primers, then RNase H2 cleavage discrimination is maximized, but primer-dimer artifacts increase and mismatch detection becomes error-prone with standard polymerases
Solution Approach 1:
The patent segments the primer into distinct functional regions: a blocked-cleavable region with RNA-DNA hybrid structure for RNase H2 cleavage, a mismatch region positioned away from the cleavage site for stable binding, and a poly-T tail for polymerase compatibility. This segmentation allows each region to perform its specific function without interfering with others, reducing primer-dimer formation while maintaining cleavage discrimination
Solution Approach 2:
The patent applies local quality by making different parts of the primer have different properties: the 5' end contains the blocked-cleavable RNA-DNA hybrid for enzyme recognition, the middle section has the mismatch position for stable hybridization, and the 3' end has poly-T for polymerase binding. Each local region is optimized for its specific function, preventing harmful interactions
3Productivity
If EMCA assays are used to detect CRISPR mutations, then the method is fast and inexpensive, but accuracy is reduced because mismatch endonuclease enzymes fail to cleave single-base events and Mut/Mut homodimers are not detected
Solution Approach 1:
The patent creates a copied detection system that mimics the EMCA workflow but uses a different molecular mechanism. Instead of using mismatch endonucleases on heteroduplexes, the patent uses blocked-cleavable primers with RNase H2 that directly detect mutations during PCR amplification, copying the efficiency benefits while avoiding the accuracy limitations
4Reliability
If blocked-cleavable primers with mismatch positioned 5' or 3' of RNA are used, then primer-dimer formation is reduced and mismatch discrimination is enhanced, but the assay design becomes more complex
Solution Approach 1:
The patent makes the blocked-cleavable primer design universal by creating a standardized structure with defined regions (blocked-cleavable region, mismatch region, poly-T tail) that can be applied to any target sequence. The universal poly-T tail and RNase H2 cleavage mechanism work across different targets, reducing the need for target-specific optimization and simplifying overall assay design despite the enhanced functionality
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 provides a more efficient and less error-prone method for detecting SNPs, indels, and mutations altered by CRISPR Cas9, while also enabling inexpensive multi-color assays and accurate visualization of multiple allele results.
Implementation Method 1
blocked-cleavable RNase H2-dependent PCR (rhPCR) primers
Implementation Method 2
high discrimination polymerase mutants
Implementation Method 3
hybridizing the primer to the target DNA sequence to form a double-stranded substrate
Data Source
AI summary
The invention can be used to provide a more efficient and less error-prone method of detecting variants in DNA, such as SNPs and indels. The invention also provides a method for performing inexpensive multiplex assays. The invention also provides methods for detection of DNA sequences altered after cleavage by a targetable endonuclease, such as the CRISPR Cas9 protein from the bacterium Streptococcus pyogenes.