Detecting Off-Target Deamination in Base Editing

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Solution Overview

Problem

Current methods for detecting off-target mutations induced by Base Editing technology are inadequate, particularly in identifying deaminated sites and spurious deamination events, due to their sensitivity and cost issues, and inability to detect mutations that occur at low frequencies or independently of site-specific targeting.

Innovation Solution

Developed methodologies include an in vitro Base-Editor targeting and deamination reaction followed by enzymatic digestion and sequencing, and a targeted PCR-based enrichment protocol using Endonuclease MS and uracil DNA glycosylase to detect deaminated sites, as well as 3D PCR to selectively amplify and sequence rare deamination-induced mutations.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If traditional sequencing methods are used to detect off-target mutations, then comprehensive coverage is achieved, but sensitivity and cost-effectiveness deteriorate

Engineering Contradiction:

Improvedetection sensitivityVSAvoidmethod complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The detection method is segmented into distinct functional modules: Base Editor deamination step, TkoEndoMS digestion step, adapter ligation step, and sequencing step. Each module performs a specific function, allowing optimization of individual steps while maintaining overall sensitivity and reducing complexity compared to comprehensive sequencing approaches.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

TkoEndoMS endonuclease acts as an intermediary that specifically recognizes and cleaves at deaminated cytosine sites (converted to uracil), creating a bridge between the chemical modification and detectable DNA fragmentation. This intermediary enables selective detection of deamination events without requiring comprehensive sequencing of the entire genome.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Measurement precision

If comprehensive sequencing is performed to detect all mutations, then detection coverage is improved, but cost and time consumption worsen

Engineering Contradiction:

Improvedetection coverageVSAvoiddetection time

Core Design Contradiction:

Measurement precisionVSLoss of time

Solution Approach 1:

The Base Editor is applied in advance to perform site-specific deamination at target loci before detection. This preliminary action concentrates the mutations of interest at specific genomic locations, allowing subsequent enrichment and detection methods to focus only on relevant sites rather than screening the entire genome, thereby reducing time and cost.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The method changes the chemical parameter of the DNA bases by converting cytosine to uracil through deamination. This parameter change creates a detectable difference between edited and unedited sites, enabling selective identification of off-target mutations without requiring comprehensive sequencing of all genomic positions.

Inventive Principle:

Principle #35Parameter changes

3Manufacturing precision

If site-specific targeting is used, then on-target efficiency is improved, but detection of spurious deamination events worsens

Engineering Contradiction:

Improvetargeting specificityVSAvoidspurious event detection

Core Design Contradiction:

Manufacturing precisionVSMeasurement precision

Solution Approach 1:

The detection method extracts and isolates deamination events from the background of unedited DNA. By using TkoEndoMS to specifically cleave at uracil-containing sites and subsequent adapter ligation, the method separates edited molecules from unedited ones, enabling detection of even rare spurious deamination events that would be obscured in comprehensive sequencing.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The method incorporates feedback through the use of control samples and replicate experiments to distinguish true spurious deamination events from artifacts. By comparing results across multiple replicates and using appropriate controls, the method provides feedback to validate detected events, ensuring that spurious events are accurately identified while maintaining confidence in on-target editing efficiency.

Inventive Principle:

Principle #23Feedback

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

These methods provide a sensitive and cost-effective means to detect and quantify off-target deamination events, improving the assessment of Base Editing technologies' mutagenic potential and enabling their safe application in therapeutic settings by identifying rare mutations and spurious deamination events.

Implementation Method 1

contacting the deaminated substrate with Endonuclease MS from Thermococcus kodakarensis (TkoEndoMS) to induce double strand breaks (DSBs) at deamination sites

Methodology Applied

Scientific EffectEnzymatic digestion: Enzyme