In Vitro DNA Library Assays for Sensitive Off-Target Site 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 CRISPR-Cas9, are limited by noise in genomic DNA assays and biased representation in random base substitution libraries, making it difficult to accurately detect and quantify off-target activities.

Innovation Solution

A method involving pre-enriched linear DNA libraries synthesized on high-density oligonucleotide arrays with unique identifiers, allowing for unbiased identification of off-target sites by enriching or screening for sequences bound, modified, or cleaved by these agents, using barcoded sequences to reconstruct modified DNA sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If genomic DNA assays are used to identify off-target sites, then comprehensive genome-wide coverage is achieved, but noise in the assays reduces detection accuracy

Engineering Contradiction:

Improvegenome-wide coverageVSAvoiddetection accuracy

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent extracts and removes the problematic genomic DNA background by using synthesized DNA libraries instead. This extraction principle eliminates the noise source while preserving the ability to comprehensively screen for off-target sites, thereby resolving the contradiction between genome-wide coverage and detection accuracy.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent creates synthetic copies of potential off-target sequences in a controlled library format. These copied sequences allow comprehensive genome-wide screening without the noise of native genomic DNA, enabling both high adaptability and measurement precision simultaneously.

Inventive Principle:

Principle #26Copying

2Adaptability or versatility

If random base substitution libraries are used to identify off-target sites, then sequence diversity is increased, but biased representation reduces identification accuracy

Engineering Contradiction:

Improvesequence diversityVSAvoididentification accuracy

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by designing libraries with controlled substitution patterns at specific positions rather than random substitutions throughout. This ensures unbiased representation of potential off-target sequences while maintaining comprehensive sequence diversity, resolving the contradiction between adaptability and identification accuracy.

Inventive Principle:

Principle #3Local quality

3Ease of operation

If conventional in vitro assays are used to detect off-target activity, then simplicity is maintained, but sensitivity is insufficient for accurate detection

Engineering Contradiction:

Improveassay simplicityVSAvoiddetection sensitivity

Core Design Contradiction:

Ease of operationVSMeasurement precision

Solution Approach 1:

The patent introduces barcoded DNA sequences as intermediaries that link the enzyme activity to detectable signals. These barcodes serve as mediators that amplify the detection signal while maintaining the simplicity of the in vitro assay format, thereby achieving both ease of operation and high detection sensitivity.

Inventive Principle:

Principle #24Intermediary (Mediator)

Data Source

PatentUS12606867B2Highly sensitive in vitro assays to define substrate preferences and sites of nucleic-acid binding, modifying, and cleaving agents

Publication Date: 2026.04.21 THE GENERAL HOSPITAL CORP

AI summary

Described herein are, among other things, methods of identifying double stranded DNA sequences that are modified by a base editing enzyme. For example, by providing a plurality of linear dsDNA oligonucleotides of known sequences, incubating the library in the presence of a base editing enzyme, amplifying the oliognucleotides with a polymerase that converts edited base pairs to equal mixtures of canonical base pairs during DNA synthesis, and determining the sequences of the amplified oligonucleotides.