Plastid Selectable Markers for Nuclear Genome Editing Selection
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Solution Overview
Problem
Current methods for nuclear genome editing in organisms like green algae are laborious and inefficient, often relying on limited nuclear selectable markers that can lead to dead-end strains and challenges with marker recycling.
Innovation Solution
The use of plastid-selectable markers to detect nuclear genome editing, where a ribonucleoprotein complex is used to transform the nucleus and a construct with a selectable marker is inserted into the plastid, allowing for selection of cells with the genetic modification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If nuclear selectable markers are used for genome editing selection, then nuclear genome modifications can be detected, but the limited number of available markers leads to dead-end strains and inefficiency
Solution Approach 1:
The patent introduces plastid selectable markers as an intermediary system to enable nuclear genome editing selection. Instead of directly selecting for nuclear marker integration, the method uses plastid markers that co-transform with the nuclear editing machinery. This intermediary approach bypasses the limitation of having few nuclear markers while maintaining the ability to select for successful nuclear genome edits.
Solution Approach 2:
The patent makes plastid selectable markers serve a dual function: they select for plastid transformation events and simultaneously serve as proxies for selecting nuclear genome editing events. This multi-functionality allows the same marker system to be used across multiple editing experiments, eliminating the need for multiple different nuclear markers and preventing dead-end strains.
2Productivity
If multiple nuclear markers are stacked to edit multiple targets, then multiple genome modifications can be achieved, but the process becomes laborious and creates dead-end strains
Solution Approach 1:
The patent enables a single plastid marker system to support multiple editing targets by co-transforming with multiple different nuclear editing constructs. Each plastid transformation event can simultaneously deliver multiple nuclear editing components, allowing multiplexed editing without requiring multiple different nuclear markers. This reduces process complexity while maintaining high editing capacity.
Solution Approach 2:
The patent combines the selection function for multiple editing events into a single plastid marker system. Instead of using separate nuclear markers for each editing target, the method merges all selection functions into the plastid marker, which co-transforms with multiple nuclear editing constructs. This consolidation simplifies the transformation process and eliminates dead-end strains caused by exhausting nuclear markers.
3Adaptability or versatility
If nuclear marker recycling is attempted using site specific recombinases, then marker reuse becomes possible, but reliable expression of additional transgenes and delivery of recombinant protein is required
Solution Approach 1:
The patent uses plastid markers as an intermediary system that naturally persists through transformation events without requiring complex recycling mechanisms. Unlike nuclear marker recycling that demands reliable expression of recombinase transgenes and delivery systems, the plastid marker approach allows markers to be maintained in the plastid genome across generations, providing inherent reusability without additional molecular machinery.
Solution Approach 2:
The plastid marker system is self-sustaining and does not require external recycling mechanisms. The markers integrate into the plastid genome and are maintained through normal plastid replication and inheritance, automatically providing reusable selection capability without needing site-specific recombinases or additional transgene expression systems.
4Ease of repair
If Cre expression or delivery is made reliable for nuclear marker recycling, then marker removal becomes possible, but aggressive non-homologous end joining and concatemer formation prevent complete marker removal
Solution Approach 1:
The patent uses plastid markers as an intermediary selection system that does not need to be removed from the organism. Since the markers are in the plastid genome rather than the nuclear genome, they can be selectively eliminated through plastid genome segregation or replacement without affecting the nuclear genome edits. This avoids the problem of incomplete marker removal that plagues nuclear marker recycling approaches.
Data Source
AI summary
The invention provides methods of using chloroplast-selectable markers to detect nuclear genome editing. The methods involve selecting a genetic modification that has occurred in a nuclear genome of a photosynthetic cell; transforming a photosynthetic cell with a ribonucleoprotein complex that effects a genetic modification in the genome in the nucleus of a photosynthetic cell; transforming the photosynthetic cell with a construct that effects an insertion of a selectable marker into the plastome of a plastid of the cell; selecting for photosynthetic cells having the selectable marker inserted into the plastome; and thereby selecting for the genetic modification to the genome in the nucleus of the photosynthetic cell.