CRISPR-Targeted Adapter Binding for Sequencing Library Normalization
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Solution Overview
Problem
Existing methods for normalizing nucleic acid concentrations between samples in next-generation sequencing are inaccurate and time-consuming, often leading to under- or over-sequencing due to variations in nucleic acid amounts.
Innovation Solution
Utilizing a catalytically inactive CRISPR-associated protein (dCas) or dArgonaute complex with a guide RNA to bind to adapter sequences in polynucleotide libraries, followed by separation and extraction to normalize concentrations between samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional methods (spectrophotometry, electrophoresis, fluorometry, qPCR) are used to detect and normalize nucleic acid concentrations, then concentration measurement is achieved, but the process is time-consuming and labor-intensive with multiple steps
Solution Approach 1:
The patent extracts the normalization function from complex multi-step detection methods and integrates it directly into the sequencing library preparation workflow. By using adapter sequences that are universally present in NGS libraries and combining them with CRISPR-based detection, the method eliminates separate concentration measurement and adjustment steps, achieving normalization as an inherent part of the library preparation process itself
Solution Approach 2:
The patent merges multiple functions into a single integrated system: adapter sequences serve both as sequencing priming sites and as targets for CRISPR-based concentration detection; the CRISPR system simultaneously detects and enables normalization of multiple samples. This consolidation eliminates the need for separate spectrophotometry, fluorometry, or qPCR steps that traditionally followed library preparation
2Measurement precision
If manual concentration adjustments are made based on traditional detection methods, then normalization is attempted, but inaccuracy is introduced in the normalization process
Solution Approach 1:
The patent implements a feedback mechanism where adapter sequences in each library sample serve as direct targets for CRISPR-based detection. The system measures actual library concentrations through CRISPR binding events and uses this feedback information to automatically adjust normalization, eliminating manual estimation errors. The CRISPR system provides real-time concentration data that directly guides the normalization process
Solution Approach 2:
The library adapters themselves serve as the detection target, making the library preparation components self-sufficient for concentration detection. The same adapter sequences used for sequencing priming also function as CRISPR targets, eliminating the need for external standards or separate detection reagents that could introduce variability
3Measurement precision
If traditional normalization methods are used, then concentration detection is achieved, but lack of sensitivity leads to under- or over-sequencing
Solution Approach 1:
The patent changes the detection parameter from bulk optical properties (absorbance, fluorescence) to molecular-specific CRISPR binding events. By targeting specific adapter sequences with high-affinity CRISPR systems, the method achieves single-molecule-level sensitivity, enabling accurate detection of low-concentration libraries that would otherwise be missed or misquantified by traditional methods
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
Achieves precise normalization of polynucleotide concentrations within 15% of each other, ensuring accurate sequencing results by adjusting for variations in sample concentrations.
Implementation Method 1
contacting a polynucleotide sequencing library with a predetermined amount of a ribonucleoprotein that binds to an adapter sequence of the polynucleotide library
Implementation Method 2
contacting the solution with a solid phase comprising an affinity tag binding molecule that is capable of binding to the affinity tag
Data Source
AI summary
Disclosed herein are methods and compositions for normalizing polynucleotide concentration. Normalizing may be accomplished using polynucleotide binding proteins (e.g., catalytically inactive CRISPR protein or catalytically inactive Argonaute proteins). The polynucleotide binding proteins may bind to the adapter sequences of a target polynucleotide. Thus, adding the same amount of a polynucleotide binding proteins to different samples and then extracting the polynucleotide protein is shown herein to extract similar amounts of target polynucleotides from the different samples.