Cas9 Loop-Inserted Base Editors for Reduced Non-Target Deamination

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Solution Overview

Problem

Existing nucleobase editors, such as CRISPR-Cas proteins combined with deaminases, suffer from undesired genome-wide spurious deamination, bystander mutations, and target proximal edits, leading to non-specific base editing events.

Innovation Solution

A fusion protein is developed with a deaminase inserted within a flexible loop of a Cas9 polypeptide, flanked by N-terminal and C-terminal fragments, which reduces off-target deamination by positioning the deaminase closer to the target nucleobase, minimizing unwanted edits.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If a deaminase is fused to the N-terminus or C-terminus of Cas9, then base editing activity is achieved, but non-target deamination and off-target effects increase

Engineering Contradiction:

Improvebase editing specificityVSAvoidnon-target deamination

Core Design Contradiction:

ReliabilityVSObject-generated harmful factors

Solution Approach 1:

The Cas9 protein is divided into N-terminal and C-terminal fragments, with the deaminase inserted between them within a flexible loop region. This segmentation isolates the deaminase activity from the terminal ends of the protein, preventing off-target deamination while maintaining targeted base editing capability.

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

A flexible loop region acts as an intermediary structure that accommodates the deaminase insertion. This loop provides a controlled environment where the deaminase can access the target base without being exposed to the entire Cas9 protein structure, thereby reducing non-specific interactions and off-target effects.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If the deaminase is inserted within a flexible loop of Cas9, then off-target deamination is reduced, but protein structure complexity increases

Engineering Contradiction:

Improveediting precisionVSAvoidprotein structure

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The flexible loop region is identified as a specific local area of the Cas9 protein that is both structurally adaptable and functionally accessible. By concentrating the deaminase insertion in this localized region rather than throughout the entire protein, the solution achieves high editing precision while minimizing overall structural complexity.

Inventive Principle:

Principle #3Local quality

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 fusion protein effectively reduces off-target deamination events, enhancing the specificity of base editing by limiting non-specific mutations, particularly within 200 bp upstream or downstream of the targeted region.

Implementation Method 1

a deaminase inserted within a flexible loop of a Cas9 polypeptide... the deaminase of the fusion protein deaminates a target nucleobase in a target polynucleotide sequence

Methodology Applied

Scientific EffectDeamination: Hydrolysis

Data Source

PatentUS12612618B2Nucleobase editors having reduced non-target deamination and assays for characterizing nucleobase editors

Publication Date: 2026.04.28 BEAM THERAPEUTICS INC

AI summary

The invention features base editors having reduced non-target deamination, methods of using the base editors, and assays for characterizing base editors as having decreased non-target deamination, e.g. compared to programmed, on-target deamination.