Self-Removing CRISPR Constructs for Clean Plant Cell Transformation

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Existing methods for removing transgenes and selectable markers from plant cells are laborious and not applicable to asexual cell propagation, posing a metabolic burden and limiting further transformations.

Innovation Solution

A nucleic acid construct with a first promoter for editing or regulating a nucleic acid target, a construct-eliminating gRNA, a CRISPR endonuclease linked to a second promoter, and multiple target sequences for the gRNA, allowing for efficient excision of the CRISPR cassette in a single transformation run.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If CRISPR-Cas9 system is used for genome editing, then editing efficiency is improved, but metabolic burden on cells increases and further transformations are limited

Engineering Contradiction:

Improvegenome editing efficiencyVSAvoidmetabolic burden

Core Design Contradiction:

Manufacturing precisionVSObject-generated harmful factors

Solution Approach 1:

The patent implements a self-removal mechanism where the CRISPR-Cas9 construct carries its own elimination instructions. After completing genome editing, the construct uses CRISPR-Cas9 to excise itself from the genome, discarding the tool once its function is fulfilled. This resolves the contradiction by removing the metabolic burden while preserving editing efficiency.

Inventive Principle:

Principle #34Discarding and recovering

Solution Approach 2:

The CRISPR-Cas9 construct performs self-elimination through an autonomous mechanism embedded within its own sequence. The construct encodes gRNA and Cas9 that target and excise the construct itself, allowing the system to service its own removal without external intervention. This self-service approach eliminates the need for additional steps to remove the tool, reducing metabolic burden while maintaining editing capability.

Inventive Principle:

Principle #25Self-service

2Reliability

If selectable markers are integrated into plant genome, then transformation selection is improved, but further applications are limited

Engineering Contradiction:

Improvetransformation selectionVSAvoidfurther transformation capability

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The selectable marker is integrated into a CRISPR-targeted construct that is designed for self-elimination. After transformation selection is completed using the marker, the CRISPR system excises the entire construct including the marker, recovering the genome to a clean state. This allows reliable initial selection while restoring adaptability for future transformations.

Inventive Principle:

Principle #34Discarding and recovering

Solution Approach 2:

The construct is designed with preliminary elimination instructions built-in from the outset. The self-removal mechanism is pre-programmed into the construct sequence, ensuring that after the marker serves its selection purpose, the construct automatically eliminates itself. This preliminary action resolves the conflict between needing the marker for selection and avoiding limitations for future work.

Inventive Principle:

Principle #10Preliminary action

3Manufacturing precision

If multiple transformation steps are used to remove transgenes, then transgene removal is achieved, but time and labor requirements increase

Engineering Contradiction:

Improvetransgene removal completenessVSAvoidremoval process duration

Core Design Contradiction:

Manufacturing precisionVSLoss of time

Solution Approach 1:

The patent merges the genome editing function and the transgene removal function into a single integrated construct. The CRISPR-Cas9 system and the self-elimination mechanism are combined in one transformation event, allowing both editing and removal to occur simultaneously rather than in separate steps. This merging dramatically reduces time and labor while achieving complete transgene removal.

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The construct enables continuous useful action by performing both genome editing and self-elimination in a single uninterrupted process. Rather than requiring discrete steps for editing followed by separate removal procedures, the system continuously executes both functions through the autonomous CRISPR-mediated excision mechanism, eliminating time losses between steps.

Inventive Principle:

Principle #20Continuity of useful action

4Productivity

If asexual propagation method is used for cell culture, then propagation efficiency is improved, but existing transgene removal methods become inapplicable

Engineering Contradiction:

Improvecell propagation efficiencyVSAvoidtransgene removal applicability

Core Design Contradiction:

ProductivityVSAdaptability or versatility

Solution Approach 1:

The self-eliminating CRISPR construct is particularly suited for asexual propagation because it does not rely on sexual recombination or segregation. The autonomous CRISPR-mediated excision mechanism works equally well in asexually propagated cells, allowing transgene removal in suspension cultures and other asexual systems where traditional segregation-based methods fail.

Inventive Principle:

Principle #25Self-service

Solution Approach 2:

The patent replaces the mechanical/biological system of sexual segregation with a molecular enzymatic system. Instead of relying on meiotic recombination and sexual propagation to separate transgenes, the system uses CRISPR-Cas9 enzymatic activity to precisely excise the transgene construct. This substitution makes the removal method applicable to asexual propagation where sexual mechanisms are absent.

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

Enables precise and efficient removal of transgenes and selectable markers from plant cells, reducing metabolic burden and facilitating further applications without leaving traces of introduced DNA.

Implementation Method 1

The CRISPR-Cas9, Cas12a (also known as Cpf1) and, recently discovered, CasX have proven to be highly efficient valuable tools for precise genome editing across a wide range of cell types and organisms.

Methodology Applied

Scientific EffectCRISPR-Cas9 genome editing: