Aspergillus Spore Color Screening via CRISPR-Cas9 Gene Knockout
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Solution Overview
Problem
Current methods for screening recombinant Aspergillus strains are inefficient due to the complex cell wall structure of Aspergillus, making visualized screening difficult, and existing methods like GFP expression require fluorescence microscopy, which is challenging, and color reaction-based methods are inaccurate and complex.
Innovation Solution
A visualized screening method using CRISPR-Cas9 gene editing to knock out spore color change-related genes (fwnA, pptA, and brnA) and genes with unchanged phenotypes (amyA, ammA, pepA, and kusA) in Aspergillus, allowing for rapid identification of recombinant strains through spore color changes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If GFP fusion method is used for visualized screening, then screening capability is provided, but device complexity increases due to requirement of fluorescence microscope and operation difficulty increases due to sorting challenge
Solution Approach 1:
The patent utilizes spore color changes as a visual marker to indicate successful gene editing. By knocking out spore color change-related genes (fwnA, pptA, brnA), the recombinant strains exhibit distinct color phenotypes that can be directly observed with the naked eye, eliminating the need for fluorescence microscopes and complex imaging equipment.
Solution Approach 2:
The patent extracts the visualized screening function from complex equipment-dependent methods and implements it through simple color-based phenotypic changes. The screening capability is transferred from requiring fluorescence microscopy to observable color differences, simplifying the overall system.
2Difficulty of detecting and measuring
If color reaction of chromogenic culture medium is used, then screening is possible, but manufacturing precision decreases due to inaccurate effect and procedure complexity increases
Solution Approach 1:
The patent employs direct spore color changes as a visual indicator of gene editing success, providing clear and accurate phenotypic differentiation. This approach eliminates the need for complex chromogenic culture media and color reaction procedures, improving both accuracy and simplicity.
3Productivity
If multiple genes are edited simultaneously, then productivity increases due to reduced screening time, but device complexity increases due to need for multiple resistance markers
Solution Approach 1:
The patent combines multiple gene editing functions into a single CRISPR-Cas9 system with multiple sgRNAs. By merging the editing of spore color change genes (fwnA, pptA, brnA) with target gene editing in one procedure, the method achieves high productivity without requiring multiple separate resistance markers or complex multi-step protocols.
Data Source
AI summary
The present disclosure discloses a visualized screening method for an Aspergillus recombinant strain with multigene editing and belongs to the technical field of gene engineering. CRISPR-Cas9 is used in the disclosure to cleave spore color change-related genes and a target gene in Aspergillus at the same time, such that editing of the target gene is visualized and an Aspergillus niger strain with multigene editing can be rapidly and efficiently screened out through spore phenotypes. Through different combinations of visualized genes and non-phenotypic change genes, rapid screening of the strain with multigene editing and simultaneous screening of multiple visualized genes are realized, and use of resistance genes in industrial strains is reduced.