Barcoded dsDNA Assays for Sensitive Off-Target Cleavage Mapping
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Solution Overview
Problem
Existing methods for identifying off-target sites of nucleic-acid binding, modifying, and cleaving agents, such as homing endonucleases, zinc fingers, TALEs, and CRISPR-Cas9 systems, are limited by noise, bias, and the inability to comprehensively assay all potential target sites, leading to inefficiencies in clinical, industrial, and research applications.
Innovation Solution
A method involving pre-enriched libraries of linear dsDNA oligonucleotides, synthesized with unique identifiers, are incubated with enzymes to identify sequences bound, modified, or cleaved, using strategies that enrich or screen for specific interactions, allowing for comprehensive identification of off-target sites through high-density oligonucleotide synthesis and next-generation sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing methods are used to identify off-target sites, then the process is simpler, but the sensitivity and comprehensiveness are insufficient due to noise and inability to assay all potential target sites
Solution Approach 1:
The patent segments the DNA library into individual oligonucleotides with unique identifiers, allowing parallel processing and comprehensive screening of all potential target sites. Each oligonucleotide can be independently assayed for enzyme interactions, enabling complete coverage of the target space without being limited by noise in bulk assays.
Solution Approach 2:
The patent adds the dimension of unique molecular identifiers to each oligonucleotide, transforming the assay from a bulk population measurement to an individually trackable system. This enables precise counting and identification of specific DNA sequences that interact with the enzyme, dramatically improving sensitivity and eliminating noise from non-specific signals.
2Reliability
If comprehensive libraries of all potential target sites are assayed, then off-target identification is more accurate, but the time and resources required increase significantly
Solution Approach 1:
The patent performs preliminary actions by synthesizing complete libraries of all potential target sites with embedded unique identifiers before the actual enzyme screening. This pre-prepared library can be directly used for high-throughput screening, eliminating the need for time-consuming library construction during the assay process and enabling rapid comprehensive evaluation.
Solution Approach 2:
The patent changes the parameter of DNA library representation from bulk population to individually barcoded molecules, enabling parallel processing and high-throughput sequencing. This parameter change allows simultaneous analysis of thousands of potential target sites in a single experiment, dramatically reducing the time required for comprehensive screening while maintaining high accuracy.
3Productivity
If bulk DNA libraries are used for screening, then the process is faster, but noise prevents accurate identification of specific off-target sites
Solution Approach 1:
The patent introduces unique molecular identifiers as intermediaries between the DNA sequence and the detection system. These identifiers act as barcodes that allow specific oligonucleotides to be tracked and identified through high-throughput sequencing, maintaining the speed of bulk processing while eliminating the noise problem by enabling precise molecular-level identification.
Solution Approach 2:
The patent replaces traditional mechanical separation and detection methods with molecular barcoding and high-throughput sequencing. Instead of physically separating individual DNA molecules for analysis, the system uses sequence-based identification through unique identifiers, enabling rapid digital counting and identification that maintains high productivity while achieving single-molecule resolution and eliminating noise.
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
Enables highly sensitive and unbiased identification of off-target DNA sequences, reducing noise and improving the accuracy of assessing enzyme specificity, applicable to a wide range of DNA-binding proteins and enzymes.
Implementation Method 1
incubating the plurality in the presence of an enzyme selected from site-specific nucleases, DNA modifying proteins, and DNA binding domains, under conditions sufficient for cleavage, modification, or binding to occur
Data Source
AI summary
Described herein are, among other things, in vitro methods of identifying double stranded DNA sequences that are cleaved by a nuclease. For example, by providing a library of defined linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a nuclease, ligating DNA adapters, amplifying cleaved oligonucleotides, and determining the sequence(s) of the amplified fragments.