Cas-gRNA RNP Genomic Library Preparation for Epigenetic Assays
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Solution Overview
Problem
Current methods for genomic library preparation and targeted epigenetic assays face challenges in efficiently characterizing epigenetic changes, particularly in chromatin accessibility and protein association with DNA loci, due to limitations in hybrid capture technology and the inability to design appropriate oligonucleotides for specific genomic regions.
Innovation Solution
The use of Cas-gRNA ribonucleoproteins (RNPs) for genomic library preparation and targeted epigenetic assays, involving the protection and modification of DNA ends, hybridization with specific sequences, and the generation of free ends for degradation and amplification, enables precise enrichment and sequencing of specific genomic regions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If hybrid capture technology is used for genomic library preparation, then DNA sequences can be enriched, but it is challenging to design appropriate oligonucleotides for specific genomic regions with unknown epigenetic features
Solution Approach 1:
The patent introduces CRISPR-Cas9 system as an intermediary tool that bridges the gap between known genomic sequences and unknown epigenetic features. The guide RNA acts as a mediator that translates sequence information into precise chromatin accessibility mapping, enabling researchers to study epigenetic marks without needing to design complex oligonucleotide probes for each specific region
Solution Approach 2:
The patent replaces the mechanical hybrid capture system with a programmable CRISPR-Cas9 system. Instead of relying on physical hybridization of oligonucleotides to capture DNA sequences, the system uses programmable guide RNAs to direct Cas9 to specific genomic locations, thereby substituting a rigid mechanical capture mechanism with a more flexible and precise molecular guidance system
2Manufacturing precision
If CRISPR-Cas9 is used for targeted chromatin accessibility mapping, then specific genomic loci can be precisely targeted, but the system requires careful optimization of guide RNA design and Cas9 activity
Solution Approach 1:
The patent segments the CRISPR-Cas9 system into modular components: guide RNA synthesis, Cas9 protein expression, and chromatin accessibility assay protocols. This segmentation allows each component to be independently optimized and standardized, reducing the overall complexity of implementing the assay while maintaining high targeting precision
Solution Approach 2:
The patent systematically varies key parameters such as guide RNA concentration, Cas9-to-guide RNA ratio, and incubation time to optimize chromatin accessibility mapping. By establishing optimal parameter ranges through preliminary experiments, the system achieves high locus targeting precision while simplifying routine assay implementation
3Quantity of substance
If genomic DNA is fragmented for sequencing library preparation, then coverage can be improved, but the fragmentation process may introduce bias and lose valuable epigenetic information
Solution Approach 1:
The patent performs chromatin accessibility mapping and epigenetic mark characterization before DNA fragmentation and sequencing library preparation. By capturing epigenetic information at the native chromatin level prior to any disruptive processing, the method preserves valuable epigenetic data while still achieving adequate sequencing coverage through subsequent standard fragmentation protocols
Solution Approach 2:
The patent extracts and analyzes chromatin accessibility and epigenetic marks as separate information layers before proceeding with standard DNA sequencing workflows. This extraction approach allows epigenetic information to be captured and preserved independently, preventing its loss during necessary DNA fragmentation steps for sequencing
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 allows for sensitive and precise characterization of epigenetic changes, improving the resolution of epigenetic assays and enabling ultra-deep analysis of fine epigenetic mechanisms, facilitating better research and clinical understanding.
Implementation Method 1
selectively generating free ends within the first double-stranded polynucleotides includes hybridizing CRISPR-associated protein guide RNA ribonucleoproteins (Cas-gRNA RNPs) to sequences that are present within the first double-stranded polynucleotides and that are not present within the second double-stranded polynucleotides
Data Source
AI summary
Genomic library preparation using Cas-gRNA RNPs, and targeted epigenetic assays, are provided herein. Some compositions include, from a first species, substantially only single-stranded polynucleotides; from a second species, substantially only double-stranded polynucleotides; and amplification primers ligated to ends of the second double-stranded polynucleotides and substantially not ligated to any ends of the first double-stranded polynucleotides. Some compositions include first and second molecules of a target polynucleotide having a sequence, the first molecule having a first end at a first subsequence, the second molecule having a first end at a second subsequence, wherein the first subsequence only partially overlaps with the second subsequence. Some examples provide a composition that includes a target polynucleotide and a first fusion protein including a Cas-gRNA RNP coupled to a transposase having an amplification adapter coupled thereto. The Cas-gRNA RNP may be hybridized to a subsequence in the target polynucleotide.