CRISPR sgRNA Pool Preparation for Sequencing Library Complexity Reduction
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current targeted enrichment methods for sequencing libraries, such as CRISPR/Cas technology, are limited in reducing the complexity of sequencing libraries and are not suitable for efficient removal of target-irrelevant sequences, leading to increased sequencing costs and data management challenges in personalized transcriptome analysis.
Innovation Solution
A method involving the purification of mRNA, conversion to cDNA, amplification, fragmentation, tagging, and the use of synthetic single guide RNAs (sgRNAs) to guide nucleic acid-binding proteins for targeted cleavage and size selection of uncut target polynucleotides, reducing the sequencing library complexity and allowing for more manageable data processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If CRISPR/Cas technology is used for targeted enrichment, then specific sequences can be cleaved, but the complexity of sequencing libraries is not sufficiently reduced
Solution Approach 1:
The invention divides the enrichment process into multiple sequential steps: (i) purifying mRNA population from sample, (ii) preparing cDNA from mRNA, (iii) amplifying target sequences from cDNA, (iv) fragmenting amplified DNA molecules, (v) connecting fragments to tag, (vi) hybridizing starting oligonucleotides with tagged catcher oligonucleotides, (vii) removing complexes by binding tag to cognate interactor, and (viii) preparing sgRNA pool with reduced starting oligonucleotide pool. This segmentation allows systematic reduction of library complexity that single-step CRISPR/Cas cannot achieve
Solution Approach 2:
The invention performs preliminary actions before the main sequencing process by pre-purifying mRNA, pre-converting to cDNA, pre-amplifying target sequences, and pre-fragmenting DNA molecules. These preliminary steps prepare the material in advance for more efficient targeted enrichment and removal of target-irrelevant sequences, reducing the burden on subsequent sequencing steps
2Loss of information
If targeted enrichment is performed to remove target-irrelevant sequences, then sequencing costs increase, but without enrichment data management becomes unmanageable
Solution Approach 1:
The invention changes key parameters of the sequencing library by reducing the total quantity of polynucleotides through selective removal of target-irrelevant sequences. By adjusting the composition and complexity of the library (changing from full transcriptome to enriched target population), the method optimizes the balance between data management requirements and sequencing costs, achieving manageable data volumes without excessive enrichment that would drive costs up
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 significantly reduces sequencing costs and data processing burdens by effectively removing target-irrelevant sequences, enabling more efficient and cost-effective next-generation sequencing.
Implementation Method 1
The Cas nuclease, when complexed with a short RNA oligonucleotide known as a single guide RNA (sgRNA), can induce double-stranded breaks (DSBs) at specific sgRNA complementary locations.
Implementation Method 2
Hybrid capture, wherein nucleic acid strands derived from the input sample are hybridized specifically to pre-prepared DNA fragments complementary to the targeted regions of interest
Data Source
AI summary
The present invention relates to a method of obtaining an enriched personalized population of a target polynucleotide using a synthetic single guide RNA (sgRNA) for an sgRNA-guided nucleic acid-binding protein, as well as to a method of obtaining a pool of personalized target-irrelevant synthetic single guide RNAs (sgRNAs) for a sgRNA-guided nucleic acid-binding protein. Also provided is a kit comprising a pool of sgRNAs obtainable by the methods of the invention, the use of a pool of sgRNAs obtainable by the methods of the invention and a method of monitoring a disease state.