Base Editor Guide RNA Arrays for Targeted Plant Mutagenesis
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Solution Overview
Problem
Existing mutagenesis techniques in plants and animals result in low mutation density, random mutations throughout the genome, and difficulty in identifying traits of interest due to the introduction of double-strand breaks and indels, making it challenging to discover and optimize agronomically important phenotypes.
Innovation Solution
Utilizing base editors with an array of guide RNAs to introduce targeted mutagenesis without double-strand breaks, allowing for high-density, trackable sequence modifications at specific genomic loci, enabling the identification of agronomically important phenotypes by screening populations for desired traits.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If chemical- or radiation-induced mutagenesis is used, then genetic variation is generated, but mutation density is low and mutations are scattered randomly throughout the genome
Solution Approach 1:
The invention segments the genome into specific target regions using guide RNAs that direct the base editor to precise locations. Instead of random mutagenesis affecting the entire genome, multiple gRNAs can be designed to target specific genes or genomic regions of interest, concentrating mutagenesis efforts where needed while leaving other regions untouched.
Solution Approach 2:
The base editor system applies mutagenesis locally to specific genomic loci rather than uniformly across the genome. By using guide RNAs with specific sequences that complement target DNA regions, the editing activity is concentrated at defined locations, creating high mutation density precisely where required while maintaining low mutation density elsewhere.
2Quantity of substance
If CRISPR/Cas9 based tools are used to introduce multiple double strand breaks, then sequence rearrangements and large deletions occur, but this completely disrupts the target locus and shuts down gene function
Solution Approach 1:
The invention extracts the DNA cleavage function from the CRISPR system by using a nickase variant (Cas9D10A) that creates single-strand nicks instead of double-strand breaks. This removes the harmful effect of complete locus disruption while retaining the ability to guide the editor to specific target sequences through the guide RNA.
Solution Approach 2:
The invention changes the parameter of DNA strand breakage from double-strand breaks (Cas9 wild type) to single-strand nicks (Cas9D10A nickase). This parameter change fundamentally alters the outcome: instead of complete locus disruption and gene shutdown, the system produces controlled base substitutions that maintain gene function while introducing desired variations.
3Object-affected harmful factors
If base editors are used to introduce direct and programmable mutations without double-stranded cleavage, then off-target effects are reduced, but the ability to introduce multiple mutations at different sites within a genetic locus is limited
Solution Approach 1:
The base editor system is designed with universal applicability across multiple genomic sites. By using a library of guide RNAs that can be exchanged or combined, the same base editor protein can be directed to any number of different target loci throughout the genome, enabling multi-site mutagenesis while maintaining the low off-target profile characteristic of base editing.
Solution Approach 2:
The system segments the mutagenesis task into multiple independent guide RNA-target pairs. Each gRNA can be designed to target a specific site within or across different genes, allowing the accumulation of multiple precise mutations at different locations without the off-target effects associated with traditional mutagenesis methods.
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
Achieves targeted mutagenesis with minimal off-target effects, enabling the discovery of novel or optimized traits in plants by introducing specific mutations at desired genomic locations, thereby improving agricultural performance.
Implementation Method 1
base editors, including BEs (base editors mediating C to T conversion) and ABEs (adenine base editors mediating A to G conversion), are powerful tools to introduce direct and programmable mutations without the need for double-stranded cleavage
Data Source
AI summary
The present invention relates to novel methods for discovering traits and generating cellular systems having improved phenotypes. In particular, the present invention provides methods for the development of plants having agronomically optimized phenotypes by using targeted mutagenesis with few or no off-target effects. Targeted mutagenesis is achieved by the introduction of a base editor complex or of a STEME complex comprising an array of guide RNAs targeting a nucleic acid sequence of interest. The present invention also relates to cellular systems obtained by the methods described herein and to the use of a base editor complex or the STEME complex comprising an array of guide RNAs for generating a cellular system having an agronomically important phenotype and for identification of an agronomically important phenotype.