CRISPR-Guided Genomic DNA Detection with Local Helicase Unwinding
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Solution Overview
Problem
The understanding of the relationship between chromosome arrangement and gene expression is limited, necessitating improved methods for detecting specific nucleic acid sequences in genomes.
Innovation Solution
A method involving a gene editing complex to induce a nick in genomic nucleic acid sequences, followed by denaturation with a helicase enzyme and detection using a detectably labeled probe complementary to the specific nucleic acid sequence, utilizing CRISPR-associated endonucleases and guide nucleic acids for precise targeting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional nucleic acid detection methods are used, then the detection process is simple, but the measurement precision and ability to detect specific sequences in complex genomes is limited
Solution Approach 1:
The detection system is segmented into distinct functional modules: a gene editing complex (Cas9 endonuclease with guide RNA) for targeted nicking, a helicase enzyme for localized denaturation, and detectably labeled probes for sequence-specific detection. This segmentation allows each component to perform its specialized function efficiently, improving measurement precision while keeping the overall system manageable through modular design.
Solution Approach 2:
The patent introduces intermediary molecules and enzymes to bridge the gap between simple detection and high precision. The guide RNA acts as an intermediary that directs the Cas9 endonuclease to specific genomic locations. The helicase serves as an intermediary that facilitates probe access by locally denaturing DNA. These intermediaries enable high detection accuracy without requiring direct complex interaction between all system components.
2Reliability
If the genome is fully denatured for probe access, then probe binding is improved, but energy consumption and potential damage to genomic DNA increase
Solution Approach 1:
Instead of uniformly denaturing the entire genome, the system applies denaturation locally only at the specific genomic regions of interest. The Cas9 endonuclease creates nicks at precise target locations, and the helicase is recruited to these specific sites to denature only the necessary DNA segments. This local quality approach ensures reliable probe binding at target sites while minimizing energy consumption and avoiding unnecessary damage to the rest of the genomic DNA.
Solution Approach 2:
The system performs preliminary targeted nicking by the Cas9 endonuclease before probe binding occurs. This preliminary action creates specific entry points that recruit the helicase to denature DNA only where needed, rather than requiring energy-intensive global denaturation. The preliminary targeted modification enables subsequent efficient probe binding with minimal energy expenditure.
3Measurement precision
If high specificity is required for mutation detection, then false positives are reduced, but the detection sensitivity and ability to detect all variants may decrease
Solution Approach 1:
The patent replaces traditional mechanical hybridization-based detection with a programmable molecular recognition system. The guide RNA sequence is programmable to match specific target sequences with high specificity. The Cas9 endonuclease provides programmable targeted nicking based on guide RNA complementarity. This substitution of mechanical binding with programmable molecular recognition enables both high specificity for mutation detection and maintained sensitivity through precise targeting of variant sequences.
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
Enables accurate detection of specific nucleic acid sequences, including mutations, in coding and non-coding regions of the genome, enhancing our understanding of chromosome arrangement and gene expression.
Implementation Method 1
inducing a nick in genomic nucleic acid sequences by a gene editing complex comprising a CRISPR-associated endonuclease and a guide nucleic acid sequence that is complementary to a target nucleic acid sequence
Implementation Method 2
denaturing the genomic nucleic acid sequences by contacting the genomic nucleic acid sequences with a helicase enzyme at the nicked genomic nucleic acid sequences
Implementation Method 3
contacting the denatured genome with a detectably labeled probe, wherein the detectably labeled probe is complementary to the specific nucleic acid sequence of interest
Data Source
AI summary
In one embodiment, methods for detecting a specific nucleic acid sequence in a genome are provided that may include: a) inducing a nick in genomic nucleic acid sequences by a gene editing complex; b) denaturing the genomic nucleic acid sequences by contacting the genomic nucleic acid sequences with a helicase enzyme at the nicked genomic nucleic acid sequences; c) contacting the denatured genome with a detectably labeled probe, wherein the detectably labeled probe is complementary to the specific nucleic acid sequence of interest; and, d) detecting the specific nucleic acid sequence of interest.