CRISPR-Cas Sequence-Specific Enrichment for Isothermal Amplification
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Solution Overview
Problem
Current methods for sequence-specific DNA enrichment and nucleic acid amplification are laborious, time-consuming, and inefficient, often requiring multiple steps and large sample amounts, and existing isothermal amplification systems lack the desired speed and efficiency for some applications.
Innovation Solution
The use of CRISPR-Cas and Argonaute systems for enriching or amplifying polynucleotides, involving the formation of complexes with target-specific nucleotide regions and subsequent hybridization and extension of primers using polymerases to achieve efficient amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current methods for sequence-specific DNA enrichment are used, then polynucleotides can be enriched, but the process is laborious, time-consuming, and requires multiple steps
Solution Approach 1:
The patent combines CRISPR-Cas system binding with isothermal amplification into a single integrated process. The CRISPR-Cas complex binds to target DNA and simultaneously initiates amplification through the recruited polymerase, eliminating the need for separate enrichment and amplification steps. This merging of functions directly resolves the contradiction by maintaining high enrichment efficiency while dramatically reducing process time.
Solution Approach 2:
The CRISPR-Cas complex performs preliminary target recognition and binding before amplification begins. The guide RNA pre-programmed with target sequence specificity allows the system to locate and bind to the exact target site, preparing the template for subsequent amplification. This preliminary action ensures high enrichment efficiency is achieved before the amplification process starts, reducing overall time required.
2Productivity
If current DNA enrichment methods are used, then target polynucleotides can be enriched, but large amounts of sample nucleic acids are required
Solution Approach 1:
The patent employs isothermal amplification conditions that allow amplification to proceed at a constant temperature, optimizing enzyme activity and reaction kinetics. This parameter change from temperature-cycling to isothermal conditions enables efficient amplification with minimal starting material. The CRISPR-Cas system further enhances this by providing sequence-specific recruitment, ensuring that even trace amounts of target DNA are efficiently enriched and amplified.
3Productivity
If temperature cycling is used for nucleic acid amplification, then amplification can be achieved, but the process requires energy and time for temperature ramp and equilibration
Solution Approach 1:
The patent replaces the mechanical temperature cycling system with a biochemical isothermal amplification system. Instead of using thermal cycles to denature and anneal DNA, the system uses a temperature-stable polymerase that functions optimally at a constant temperature. The CRISPR-Cas complex recruits this polymerase to the target site, and amplification proceeds through isothermal conditions, eliminating the need for energy-intensive temperature cycling while maintaining high amplification efficiency.
4Loss of time
If isothermal amplification methods are used, then energy and time are saved, but the desired speed and efficiency are not achieved
Solution Approach 1:
The CRISPR-Cas complex serves as an intermediary that dramatically enhances isothermal amplification speed and efficiency. The guide RNA-programmed Cas protein specifically binds to target DNA sequences and recruits the polymerase directly to the target site. This intermediary function concentrates amplification activity at the exact location needed, accelerating the reaction rate and achieving high productivity under isothermal conditions without requiring temperature cycling.
5Productivity
If additional enzymes and reagents are used for isothermal amplification, then amplification can proceed, but the system becomes more complex and costly
Solution Approach 1:
The CRISPR-Cas system provides multi-functionality by combining target recognition, binding, and amplification initiation in a single system. The guide RNA provides sequence specificity, the Cas protein provides target binding and recruitment, and the polymerase provides amplification. This universal system eliminates the need for separate enrichment reagents and multiple specialized enzymes, reducing overall system complexity and cost while maintaining high amplification 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
Provides rapid and efficient sequence-specific polynucleotide enrichment and amplification, reducing the need for temperature cycling and minimizing the use of additional enzymes and reagents, suitable for applications like next-generation sequencing.
Implementation Method 1
a clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA) or a derivative thereof, and a CRISPR-associated (Cas) protein or a variant thereof, wherein the crRNA or the derivative thereof contains a target-specific nucleotide region complementary to a region of a first strand of the target double-stranded nucleic acid
Implementation Method 2
extending a nucleic acid complementary to the second strand of the target double-stranded nucleic acid from the primer using a polymerase
Data Source
AI summary
A method for enriching or amplifying a target nucleic acid including providing a system having a guide nucleic acid, and a Cas or Argonaute protein or a variant thereof. The guide nucleic acid contains a target-specific nucleotide region substantially complementary to a region of the target nucleic acid, and contacting the target nucleic acid with the system to form a complex.