Monocot Genome Editing via Base Conversion Complex

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Solution Overview

Problem

Current genome editing techniques for monocots, such as those using CRISPR-Cas9, often require double-stranded DNA breaks, leading to cytotoxicity, chromosomal translocations, and low viable cell numbers, making efficient genetic modification difficult.

Innovation Solution

A method involving a complex of a nucleic acid sequence-recognizing module and a deaminase, where the deaminase is fused with a Cas9 protein optimized for monocots, and a nuclear localization signal is added to enhance nuclear translocation, allowing for nucleotide conversion without DNA cleavage, and culturing at lower temperatures to improve mutation introduction efficiency.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If conventional genome editing techniques (CRISPR-Cas9, ZFN, TALEN) are used to modify monocot genomes, then sequence-specific DNA cleavage and mutation introduction are achieved, but cytotoxicity increases, chromosomal translocations occur, and viable cell numbers decrease

Engineering Contradiction:

Improvegenome editing precisionVSAvoidcytotoxicity and chromosomal translocation

Core Design Contradiction:

Manufacturing precisionVSObject-affected harmful factors

Solution Approach 1:

The invention extracts and removes the DNA cleavage function from the genome editing system. By using deaminases that catalyze base conversion without causing DNA strand breaks, the harmful effects of cleavage (cytotoxicity, chromosomal translocation) are eliminated while retaining the ability to introduce precise mutations at target sites

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention replaces the mechanical DNA cleavage mechanism with a chemical base conversion mechanism. Instead of using nucleases to physically cut DNA strands, deaminases chemically convert bases (e.g., cytosine to uracil), achieving mutation introduction without mechanical disruption of the DNA backbone

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Manufacturing precision

If DNA double-stranded breaks are induced for genome editing, then mutation introduction is achieved, but the number of viable cells becomes extremely small and genetic modification becomes difficult

Engineering Contradiction:

Improvemutation introduction efficiencyVSAvoidviable cell number

Core Design Contradiction:

Manufacturing precisionVSQuantity of substance

Solution Approach 1:

The invention converts the previously harmful DNA cleavage step into a beneficial base conversion process. By using deaminases to directly convert bases at target sites without causing strand breaks, the system achieves mutation introduction while preserving cell viability, thus converting what was once a harmful side effect into the primary mechanism of action

Inventive Principle:

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

3Productivity

If Agrobacterium method is used for monocot transformation, then efficient gene transfer is achieved, but monocots are not natural hosts and require optimization

Engineering Contradiction:

Improvetransformation efficiencyVSAvoidhost compatibility

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The invention creates a genome editing system with broad universality across monocot species. By using deaminases combined with guide RNAs that can target any DNA sequence, the system achieves species-independent functionality, working efficiently in rice, wheat, maize and other monocots without requiring species-specific optimization

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 safe and efficient genetic modification of monocots with high mutation introduction efficiency, reducing cytotoxicity and off-target effects by avoiding DNA breaks, and allowing for precise nucleotide substitutions or deletions.

Implementation Method 1

a deaminase catalyzing a deamination reaction is linked to a molecule having a DNA sequence recognizing ability

Methodology Applied

Scientific EffectDeamination reaction:

Data Source

PatentEP3382019B1Method for converting monocot plant genome sequence in which nucleic acid base in targeted DNA sequence is specifically converted, and molecular complex used therein

Publication Date: 2022.05.04 KOBE UNIV

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AI summary

The present invention provides a method of modifying a targeted site of a double stranded DNA of a monocot cell, comprising a step of contacting a complex wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in the given double stranded DNA and a nucleic acid base converting enzyme are bonded, with said double stranded DNA, to convert one or more nucleotides in the targeted site to other one or more nucleotides or delete one or more nucleotides, or insert one or more nucleotides into said targeted site, without cleaving at least one strand of said double stranded DNA in the targeted site, wherein the double stranded DNA is contacted with the complex by introducing a nucleic acid encoding the complex into the monocot cell. Furthermore, also provided is a complex used for the method, wherein a nucleic acid sequence-recognizing module that specifically binds to a target nucleotide sequence in a double stranded DNA of a monocot cell and a nucleic acid base converting enzyme are bonded.