Cas9 Base Editing for Low-Genotoxicity Lymphohematopoietic Engineering

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Solution Overview

Problem

Current methods for genetic engineering of human immune cells, such as using CRISPR systems for gene editing, face challenges with inefficiency in precision alterations, high toxicity due to double-strand breaks, and potential oncogenic risks.

Innovation Solution

The use of base editor fusion proteins, comprising a deaminase domain fused to a Cas9 nickase domain with a base excision repair inhibitor domain, in conjunction with splice acceptor-splice donor gRNAs, to modify lymphohematopoietic cells, allowing for precise genetic modifications with reduced toxicity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If CRISPR systems are used to induce double-strand breaks for gene editing, then gene disruption efficiency is improved, but genotoxicity and oncogenic risks increase

Engineering Contradiction:

Improvegene disruption efficiencyVSAvoidgenotoxicity

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent segments the DNA cutting function into two separate single-strand nicks instead of one double-strand break. By using two separate gRNAs that each create a single-strand nick, the system achieves gene disruption without the harmful effects of double-strand breaks, thereby reducing genotoxicity while maintaining editing efficiency

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent introduces a base excision repair inhibitor as an intermediary component that blocks the cell's normal repair mechanisms. This inhibitor forces the cell to use alternative repair pathways that are less error-prone, reducing the formation of harmful mutations and genotoxicity while still achieving the desired genetic modification

Inventive Principle:

Principle #24Intermediary (Mediator)

2Adaptability or versatility

If multiple double-strand breaks are induced for multiplexed gene editing, then genetic engineering versatility is improved, but formation of gross chromosomal translocations increases

Engineering Contradiction:

Improvemultiplexed gene editing capabilityVSAvoidchromosomal translocations

Core Design Contradiction:

Adaptability or versatilityVSObject-generated harmful factors

Solution Approach 1:

The patent applies segmentation by dividing each gene targeting event into two separate single-strand nicking events rather than one double-strand break. When performing multiplexed editing, this segmentation approach across multiple targets dramatically reduces the cumulative risk of chromosomal translocations while maintaining the ability to edit multiple genes simultaneously

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent converts the potentially harmful double-strand break mechanism into a beneficial single-strand nicking approach. By using base excision repair inhibitors with nickase Cas9, the system transforms what would normally be a high-risk operation into a safe editing method that achieves multiplexed gene disruption without generating chromosomal translocations

Inventive Principle:

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

3Manufacturing precision

If homology-directed repair is used for precise nucleotide alterations, then precision is improved, but editing efficiency decreases

Engineering Contradiction:

Improvenucleotide alteration precisionVSAvoidediting efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent substitutes the mechanical homology-directed repair process with a biochemically-driven base editing mechanism. By using deaminase enzymes that directly chemically convert bases (e.g., C to U, then to T), the system achieves precise nucleotide alterations without requiring the complex and inefficient homology-directed repair machinery, thereby improving editing efficiency while maintaining precision

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

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

This approach enables efficient and controlled multiplexed genetic engineering of human immune cells, achieving high conversion efficiencies of target bases with reduced off-target effects and genotoxicity, thereby enhancing the safety and precision of gene editing.

Implementation Method 1

a plasmid, mRNA, or protein encoding a base editor fusion protein comprising a deaminase domain fused to a Cas9 nickase domain

Methodology Applied

Scientific EffectDeamination:

Implementation Method 2

wherein the nickase domain comprises a base excision repair inhibitor domain

Methodology Applied

Scientific EffectBase excision repair inhibition:

Data Source

PatentUS20250290101A1Lymphohematopoietic engineering using cas9 base editors

Publication Date: 2025.09.18 REGENTS OF THE UNIVERSITY OF MINNESOTA

AI summary

Provided herein are methods and systems for targeted gene disruption (knock-out, missense mutation) and targeted gene knock-in in mammalian cells using base editors and guide RNAs (gRNAs) designed to target splice acceptor-splice donor sites. Also provided herein are universally acceptable genetically engineered cells comprising targeted disruptions in immunotherapy-related genes and comprising a CAR/TCR for therapeutic applications.