CRISPR Off-Target Mapping via ssDNA Enrichment
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Solution Overview
Problem
Current methods for mapping CRISPR off-target sites are either expensive, have low throughput, or require complex and laborious protocols, making it difficult to accurately identify unintended effects of CRISPR-based reagents, especially in clinical contexts.
Innovation Solution
A method involving contacting an RNA-guided endonuclease with target DNA to produce single-stranded DNA, modifying unpaired guanine bases with a chemoselective group, linking a specific binding member to this group, fragmenting the DNA, and enriching for specific binding pairs to identify off-target sites, which can be sequenced using next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If deep whole-genome sequencing is used to map off-target effects, then measurement precision is improved, but cost increases significantly
Solution Approach 1:
The patent extracts only the relevant information (off-target sites) from the entire genome by using guide RNA to specifically target and enrich for off-target locations. This allows accurate off-target mapping without requiring deep sequencing of the entire genome, thus reducing cost while maintaining precision.
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that binds to off-target sites and facilitates their enrichment. This mediator enables selective capture of off-target sites from the complex genomic background, allowing accurate mapping with reduced sequencing depth requirements.
2Measurement precision
If direct experimental mapping methods (BLESS, GUIDE-seq, HTGTS) are used to map off-target sites, then measurement precision is improved, but device complexity and protocol difficulty increase
Solution Approach 1:
The patent uses a universal approach where guide RNA serves multiple functions: it guides the endonuclease to target sites, creates the ssDNA structure, and enables enrichment of off-target sites. This multi-functionality simplifies the overall protocol by reducing the need for multiple separate reagents and steps.
Solution Approach 2:
The guide RNA itself creates the structural feature (ssDNA) that enables its own detection and enrichment. The system uses the guide RNA's binding activity to generate the signal for off-target identification, eliminating the need for separate labeling or detection reagents.
3Productivity
If ChIP-seq is used to map catalytic dead dCas9 occupancy sites, then productivity is improved, but measurement precision deteriorates due to background and specificity issues
Solution Approach 1:
The patent applies a local quality approach by focusing the enrichment strategy specifically on sites where guide RNA creates ssDNA structures. This localized enrichment at ssDNA-containing regions improves specificity by excluding background signals from other genomic regions, while maintaining high throughput through parallel processing.
Solution Approach 2:
The patent changes the detection parameter from protein occupancy (as in ChIP-seq) to ssDNA structure formation. This parameter change allows for more specific detection of actual guide RNA binding events, improving measurement precision while maintaining productivity through efficient enrichment and sequencing protocols.
4Measurement precision
If long-read sequencing methods (SMRT-OTS, Nano-OTS) are used to profile Cas9 specificity, then measurement precision is improved, but productivity decreases and cost increases
Solution Approach 1:
The patent segments the genome into potential off-target regions by using guide RNA to enrich for sites with specific ssDNA structures. This segmentation allows for targeted sequencing of only the relevant regions rather than requiring expensive long-read sequencing of the entire genome, thus improving productivity while maintaining precision.
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 provides a faster, more accessible, and versatile method for mapping CRISPR off-target sites, enabling accurate identification of potential unintended effects with moderate sequencing depth and applicability to both in vitro and in vivo settings.
Implementation Method 1
contacting the RNA-guided endonuclease with a target double-stranded DNA, thereby producing single stranded DNA (ss-DNA) at the site to which the guide-RNA binds
Implementation Method 2
reacting the product of step (a) with or an analog thereof modified by a chemoselective group, thereby adding the chemoselective group to any unpaired guanine base within the ssDNA
Implementation Method 3
linking a first binding member of a specific binding pair to the chemoselective group added in step (b)
Implementation Method 4
enriching for fragments that contain the first binding member of the specific binding pair by binding the product of step (d) to a support that comprises a second binding member of the specific binding pair, which specifically binds to the first binding member of the specific binding pair
Data Source
AI summary
The disclosure pertains to a method for isolating DNA fragments that contain a binding site for a guide-RNA of an RNA-guided endonuclease. The method comprises contacting the RNA-guided endonuclease with a target double-stranded DNA, thereby producing a single stranded DNA (ss-DNA) at the site to which the guide-RNA binds. The ssDNA is then reacted with kethoxal or an analog thereof modified by a chemoselective group, thereby adding the chemoselective group to any unpaired guanine based within the ssDNA. A first binding member of a specific binding pair is linked to the chemoselective group, for example, via a cycloaddition reaction. The DNA is fragmented and the DNA fragments that contain the binding sites for the guide-RNA are enriched using a support that comprises a second binding member of the specific binding pair. The method can be used to identify binding sites for a guide-RNA thereby identifying off-target binding sites.