Base-Editing Systems with Non-Protein UDG Inhibitors

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Solution Overview

Problem

Current genome engineering tools, such as Cas9 base-editing systems, face limitations in targeting certain genomic regions due to dependency on protospacer adjacent motifs (PAM) and suffer from incomplete uracil-DNA glycosylase (UDG) inhibition, leading to reduced editing efficiency and off-target alterations.

Innovation Solution

A base-editing system comprising a fusion protein with a DNA-binding domain, a cytidine deaminase domain, and a non-protein uracil-DNA glycosylase inhibitor (npUGI) is developed, which includes various DNA-binding domains like Cas, TALE, or Zinc finger domains, and improves UDG inhibition for enhanced editing efficiency across a wider range of sequences.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If Cas9 base-editing systems are used, then DNA editing capability is achieved, but targeting capability is limited due to PAM dependency

Engineering Contradiction:

Improvetargeting capabilityVSAvoidediting efficiency

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent changes the PAM recognition parameter by using different Cas variants (Cas12a, Cas12h, Cas12i, CasX, CasY) that recognize different PAM sequences, and by engineering mutated Cas9 variants with altered PAM specificities. This allows the system to target previously inaccessible genomic regions while maintaining editing functionality.

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent creates a universal base-editing platform that can function across diverse genomic contexts by combining multiple Cas variants with different PAM specificities. The system can adapt to various PAM sequences (e.g., TTTV for Cas12a, ATTV for Cas12h, CTTV for Cas12i, AT for CasX/Y), making it universally applicable to different target sites in the genome.

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Productivity

If protein uracil-DNA glycosylase inhibitors are used, then base-editing efficiency is improved, but incomplete UDG inhibition occurs leading to off-target alterations

Engineering Contradiction:

Improvebase-editing efficiencyVSAvoidoff-target alterations

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent introduces a non-protein UDG inhibitor as an intermediary molecule that mediates the inhibition of uracil-DNA glycosylase. This small molecule inhibitor acts as a bridge between the base editor and the UDG enzyme, providing more complete and specific inhibition compared to protein-based inhibitors, thereby reducing off-target effects while maintaining high editing efficiency.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the chemical nature of the UDG inhibitor from protein-based to non-protein (small molecule) based. This parameter change in the inhibitor's molecular structure enables more complete UDG inhibition with higher specificity, preventing the incomplete inhibition and subsequent off-target alterations that occur with protein-based inhibitors.

Inventive Principle:

Principle #35Parameter changes

3Productivity

If current genome engineering tools are used, then DNA cleavage is achieved, but off-target gene alterations occur

Engineering Contradiction:

Improveediting efficiencyVSAvoidoff-target alterations

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent extracts the cleavage function from the base-editing system by using nickase variants (Cas9n, Cas12an) that perform single-strand nicks instead of double-strand breaks. This extraction of the harmful cleavage activity while retaining the base modification capability significantly reduces off-target alterations while maintaining productive editing through the nick-induced repair pathway.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent converts the potentially harmful off-target cleavage activity into a beneficial single-strand nicking mechanism. The nick induces a controlled repair response that facilitates precise base editing without causing the harmful double-strand breaks and large indels associated with traditional nucleases, thereby transforming a harmful effect into a useful editing mechanism.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

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

The system achieves higher efficiency in editing target nucleotide sequences with reduced off-target alterations, enabling precise and reliable genome editing in medical, industrial, and research applications.

Implementation Method 1

a cytidine deaminase domain

Methodology Applied

Scientific EffectDeamination:

Implementation Method 2

a non-protein uracil-DNA glycosylase inhibitor (npUGI)

Methodology Applied

Scientific EffectEnzyme inhibition:

Data Source

PatentUS20230002746A1Base-editing systems

Publication Date: 2023.01.05 INARI AGRICULTURE TECHNOLOGY INC

AI summary

The present disclosure relates to base-editing systems including a fusion protein including a DNA-binding domain and a cytidine deaminase domain and a non-protein uracil-DNA glycosylase inhibitor, and methods of using the same. The DNA-binding domains of base-editing systems of the present disclosure include domains with a variety of target region possibilities, which increase the number and type of sequences that can be edited. The npUGIs of the base-editing systems of the present disclosure improve UDG inhibition (e.g., UDG inhibition is more complete) and are suitable for use in a wide range of organisms.