CRISPR-Cas9 Targeted Modification of GC-Rich Microorganisms
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current genetic engineering methods for GC rich microorganisms, such as oleaginous yeasts with high GC content, are limited by random gene insertion, off-target effects, and the dominance of non-homologous end joining over homologous recombination, making targeted and efficient genetic modification challenging.
Innovation Solution
A method involving the use of a RNA-guided endonuclease, such as CRISPR-associated Cas endonucleases, combined with guide RNAs and optional donor DNA, to specifically modify GC rich microorganisms. This method includes pretreating the microorganisms to degrade their cell walls, allowing for efficient transformation and targeted gene integration.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Agrobacterium mediated transformation is used for stable random integration of expression cassettes into the genome of GC rich microorganisms, then stable gene integration is achieved, but gene expression levels become unpredictable and random
Solution Approach 1:
The CRISPR-Cas system performs preliminary targeted cleavage of the genome at specific locations before integration occurs. By pre-defining the integration site through guide RNA-directed Cas9 cleavage, the system ensures that subsequent donor DNA integration happens at a predetermined location, enabling controlled and predictable gene expression while maintaining stable integration
Solution Approach 2:
The invention introduces an intermediary mechanism (CRISPR-Cas system with guide RNA) that mediates between the random integration process and the desired targeted integration. This intermediary system directs the integration event to specific genomic locations through sequence-specific recognition, thereby controlling gene expression levels while achieving stable integration
2Productivity
If non-homologous end joining (NHEJ) pathway is dominant in GC rich microorganisms, then rapid DNA repair occurs, but targeted gene modification becomes difficult
Solution Approach 1:
The CRISPR-Cas system applies local quality by creating site-specific double-strand breaks at precisely defined locations in the genome. This localized cleavage event directs the cellular repair machinery to specific sites, enabling targeted modification while utilizing the cell's inherent DNA repair pathways. The local action of Cas9 at specific genomic coordinates overrides the random nature of dominant NHEJ
Solution Approach 2:
The invention changes the parameter of DNA repair targeting from random (NHEJ-dominated) to specific (CRISPR-directed). By introducing guide RNA sequences that complement target genomic regions, the system alters the spatial parameter of repair activity, directing NHEJ or HDR processes to predetermined locations for precise gene modification
3Stability of the object's composition
If high GC content and repetitive sequences are present in GC rich microorganisms, then genomic stability is maintained, but off-target effects and nucleic acid secondary structure formation increase
Solution Approach 1:
The CRISPR system incorporates feedback mechanisms through guide RNA design and validation. By selecting guide sequences that specifically match target sites and avoiding regions with high GC content or repetitive sequences, the system provides negative feedback against off-target binding. The guide RNA acts as a feedback-controlled element that enhances on-target specificity while minimizing interactions with stable but non-target genomic regions
4Ease of manufacture
If random gene insertion is used in GC rich microorganisms, then genetic modification is achieved, but productivity and product diversity are restricted
Solution Approach 1:
The CRISPR-Cas system performs preliminary targeted cleavage at specific genomic locations before integration. By pre-defining multiple potential integration sites through guide RNA design, the system enables subsequent insertion of diverse genetic elements at controlled locations, thereby enhancing product diversity while maintaining ease of manufacture through a standardized protocol
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
The method enables precise and efficient genetic modification of GC rich microorganisms, reducing off-target effects and allowing for the targeted production of specific compounds like acetyl-CoA-based hydrophobic compounds and oils with tailored fatty acid profiles.
Implementation Method 1
A method involving the use of a RNA-guided endonuclease, such as CRISPR-associated Cas endonucleases, combined with guide RNAs and optional donor DNA, to specifically modify GC rich microorganisms
Implementation Method 2
This method includes pretreating the microorganisms to degrade their cell walls, allowing for efficient transformation and targeted gene integration
Data Source
AI summary
The present invention relates to a method of genetically modifying a GC rich microorganism. The present invention further relates to a genetically modified GC rich microorganism. Furthermore, the present invention relates to a composition comprising a RNA-guided endonuclease, at least one guide RNA (gRNA), and optionally donor DNA. The present invention also relates to a method of preparing a target compound, e.g. an acetyl-CoA-based hydrophobic compound, and/or an oil having a specific fatty acid profile, e.g. high oleic oil, using a genetically modified GC rich microorganism.