Targeted Nucleic Acid Enrichment for Genome-Wide Off-Target Detection
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Solution Overview
Problem
Current methods for detecting genome-wide gene editing off-target effects are inefficient, require high sample input, are time-consuming, and lack consistent sensitivity and specificity, especially in in vivo applications, and struggle with enriching unknown sequences contiguous to target sequences.
Innovation Solution
A method involving the use of universal oligonucleotide adaptors to ligate to single-strand nucleic acid fragments, followed by nested PCR amplification steps to enrich and identify target nucleic acids, allowing for the formation of sequencing libraries and mapping to a reference genome to evaluate gene editing efficiency and identify off-target sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If multiplex target enrichment using forward and reverse primers is employed, then target sequences can be enriched, but unknown sequences contiguous to the target sequences cannot be enriched
Solution Approach 1:
The patent introduces universal oligonucleotide adaptors as intermediary molecules that ligate to the 5' ends of target and off-target sequences. These adaptors serve as universal binding sites that enable enrichment of unknown sequences without requiring prior knowledge of their sequences, thereby resolving the contradiction between detection sensitivity and enrichment coverage
Solution Approach 2:
The patent performs preliminary ligation of universal adaptors to nucleic acid fragments before PCR amplification. This preliminary action prepares all sequences (including unknown off-targets) for subsequent enrichment, allowing comprehensive detection without requiring separate primer designs for each target
2Productivity
If forward and reverse primers are used for target enrichment, then target sequences can be amplified, but data analysis becomes challenging due to identical start and end positions
Solution Approach 1:
The patent creates asymmetric enrichment by using universal adaptors ligated to 5' ends combined with nested primers that have different binding positions. This asymmetry in primer binding positions (one universal, one nested) simplifies data analysis by providing distinct start and end positions for all enriched sequences, eliminating the ambiguity of identical boundaries
Solution Approach 2:
The patent segments the enrichment process into two distinct PCR steps with different primer combinations. The first PCR uses universal adaptor primers and target-specific primers, while the second PCR uses nested primers. This segmentation allows for simplified data analysis by creating products with predictable and distinct boundaries
3Measurement precision
If current off-target profiling methods are used, then off-target detection can be performed, but sample input requirements are high
Solution Approach 1:
The patent employs nested PCR where the second set of primers binds within the amplicons generated by the first PCR. This nested approach provides exponential amplification efficiency, enabling sensitive off-target detection from low input samples while maintaining high detection accuracy through the specificity of nested primer binding
4Measurement precision
If current off-target profiling methods are used, then off-target detection can be performed, but the process is time-consuming
Solution Approach 1:
The patent implements a continuous two-PCR protocol where the first PCR generates initial amplicons and the second nested PCR immediately amplifies these products. This continuous process without intermediate purification steps maintains detection sensitivity while significantly reducing total profiling time compared to traditional methods that require separate validation steps
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
Enhances the sensitivity and specificity of off-target detection, reduces sample requirements, and enables efficient identification of genome-wide off-target sites and evaluation of gene editing efficiency, particularly in complex samples like CRISPR-edited cells.
Implementation Method 1
contacting a universal oligonucleotide adaptor with the sample to produce a ligation product, wherein the universal oligonucleotide adaptor is configured for ligating to a 5′ end of the single-strand nucleic acid fragments
Implementation Method 2
amplifying the ligation product by a first PCR with a first target-specific primer and optionally a first universal oligonucleotide adaptor primer to form a first PCR product
Implementation Method 3
amplifying the first PCR product by a second PCR with a second target-specific primer and a second universal oligonucleotide adaptor primer to form a second PCR product
Data Source
AI summary
The present disclosure relates to enriching nucleic acid from a sample. In some embodiments, the present disclosure provides methods for enriching at least one targeted nucleic acid, identifying genome-wide gene editing off-targets, and evaluating gene editing efficiency from a sample comprising a plurality of single-strand nucleic acid fragments. Others example embodiments are also described herein.