SWISS Multiplex Genome Editing System for Plant Trait Stacking

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Solution Overview

Problem

Current multiplex genome editing technologies face challenges in plants, particularly crops, due to low efficiency of homologous recombination and limitations in delivering multiple CRISPR systems, making it difficult to stack agronomic traits or modify gene regulation networks effectively.

Innovation Solution

A CRISPR nickase-based system, SWISS (Simultaneous and Wide-editing Induced by Single System), utilizing nCas9 and scaffold RNAs with RNA aptamers to recruit cytosine and adenine deaminase proteins, enabling simultaneous CBE, ABE, and DSB at different target sites in a single transformation.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If multiple CRISPR systems are used to achieve multiplex genome editing in plants, then editing functionality is improved, but delivery complexity and system complexity increase

Engineering Contradiction:

Improvemultiplex editing functionalityVSAvoidsystem complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent combines multiple CRISPR systems (Cas9, CBE, ABE) into a single integrated SWISS system that can perform multiplex editing functions through one transformation event, reducing delivery complexity while maintaining versatile editing capabilities

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The SWISS system is designed as a universal platform that can simultaneously perform different types of genome editing (DSB, CBE, ABE) at different target sites using a single transformation, making the system multi-functional and adaptable to various editing needs

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Manufacturing precision

If homologous recombination is used for gene editing in plants, then precise editing is achieved, but editing efficiency remains low

Engineering Contradiction:

Improveediting precisionVSAvoidediting efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The patent modifies the editing approach by changing from relying solely on homologous recombination to using CRISPR-induced DSBs that leverage the plant's endogenous repair mechanisms, thereby improving editing efficiency while maintaining precision through targeted DSB formation

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent introduces CRISPR/Cas system as an intermediary that creates controlled DNA breaks, which then serve as intermediaries for the cell's repair machinery to incorporate desired edits, effectively bridging the gap between precision and efficiency

Inventive Principle:

Principle #24Intermediary (Mediator)

3Productivity

If single transformation is used for genome editing, then delivery efficiency is improved, but achieving multiplex editing at multiple sites becomes difficult

Engineering Contradiction:

Improvedelivery efficiencyVSAvoidmultiplex editing capability

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The patent merges multiple editing functions (Cas9, CBE, ABE) and multiple target sites into a single transformation event, allowing simultaneous multiplex editing at different locations in the genome through one delivery process

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The patent segments the editing functions into different modular components (Cas9 protein, CBE protein, ABE protein, sgRNAs) that can be independently designed and delivered together in a single transformation, enabling flexible multiplex editing configurations

Inventive Principle:

Principle #1Segmentation

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

SWISS achieves efficient multiplex genome editing in plants by enhancing C-to-T and A-to-G base editing frequencies and inducing deletions, facilitating the stacking of agronomic traits and modifying gene regulation networks with improved precision and efficiency.

Implementation Method 1

SpCas9 may be engineered into a nickase nCas9 (Nickase Cas9) by replacing aspartic acid at position 10 (Asp10) or histidine at position 840 (His840) with alanine (Ala)

Methodology Applied

Scientific EffectNickase activity: Enzyme

Implementation Method 2

employing two kinds of scRNAs containing different RNA aptamers to recruit corresponding RNA aptamer-binding proteins fused with cytosine deaminase or adenine deaminase

Methodology Applied

Scientific EffectCytosine deamination: Enzyme

Implementation Method 3

employing two kinds of scRNAs containing different RNA aptamers to recruit corresponding RNA aptamer-binding proteins fused with cytosine deaminase or adenine deaminase

Methodology Applied

Scientific EffectAdenine deamination: Enzyme

Implementation Method 4

two kinds of scRNAs containing different RNA aptamers to recruit corresponding RNA aptamer-binding proteins

Methodology Applied

Scientific EffectRNA aptamer binding:

Implementation Method 5

Cas9 is used to generate a double strand break (DSB) in a genome

Methodology Applied

Scientific EffectCas9 nuclease activity: Enzyme

Data Source

PatentUS20240117368A1Multiplex genome editing method and system

Publication Date: 2024.04.11 SUZHOU QI BIODESIGN BIOTECHNOLOGY CO LTD

AI summary

The invention relates to the field of plant genetic engineering. In particular, the invention relates to a method and system for multiplex genome editing suitable for plants, especially crops. More particularly, the invention relates to a CRISPR nickase-based system and method, which can simultaneously carry out different types of genome editing.