Two-Step Gene Targeting Method for Precise Yeast Mutant Identification
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Solution Overview
Problem
Gene targeting efficiency in non-conventional yeasts and filamentous fungi is low due to dominance of the non-homologous end-joining pathway, making it challenging to achieve precise modifications and identify disruption mutants, especially in organisms like Pichia pastoris where conventional methods result in low homologous recombination frequencies and random integration.
Innovation Solution
A two-step gene targeting method using a nucleotide construct with a target gene active variant, site-specific recombination sites, and a recombinase to excise the targeting cassette, allowing for precise integration and identification of disruption mutants by altering the cellular phenotype, thereby enhancing gene targeting efficiency and simplifying the identification of disruption strains.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional gene targeting methods are used in non-conventional yeasts, then random integration occurs frequently, but precise gene replacement efficiency is low
Solution Approach 1:
The patent applies preliminary action by first integrating a targeting cassette with selectable marker and site-specific recombination sites into the target gene locus through homologous recombination, creating a conditional disruption state. The actual gene disruption is then achieved in a second step by expressing recombinase to excise the cassette, leaving behind a precise modification. This two-step preliminary action resolves the contradiction by enabling precise gene replacement while maintaining high selection efficiency at each stage.
Solution Approach 2:
The patent uses an intermediary targeting cassette containing site-specific recombination sites (loxP or FRT) and selectable markers as a mediator between the wild-type gene and the final disrupted gene. This intermediary structure allows the gene to be temporarily disrupted for selection purposes, then precisely modified through recombinase-mediated excision. The intermediary resolves the contradiction by providing a selectable intermediate state that enables precise final modification.
2Manufacturing precision
If Ku70 deletion is used to reduce random integration, then homologous recombination efficiency increases, but cellular fitness and DNA repair capability are compromised
Solution Approach 1:
The patent segments the gene disruption process into two independent steps: (1) integration of targeting cassette via homologous recombination, and (2) excision of cassette via site-specific recombination. This segmentation allows the use of wild-type Ku70-containing cells for both steps, avoiding the need for Ku70 deletion. The segmentation resolves the contradiction by enabling high homologous recombination efficiency without compromising cellular fitness or DNA repair capability.
Solution Approach 2:
The targeting cassette acts as an intermediary that mediates gene disruption through recombinase excision rather than requiring Ku70 deletion. The cassette contains selectable markers for selection and site-specific recombination sites for precise excision. This intermediary approach resolves the contradiction by achieving high homologous recombination efficiency in wild-type cells without compromising cellular fitness or DNA repair function.
3Measurement precision
If extensive screening is performed to identify disruption mutants, then accurate identification is achieved, but time and resource consumption increase
Solution Approach 1:
The patent employs colorimetric screening markers (such as β-glucuronidase or alkaline phosphatase) that produce visible color changes when expressed. Disruption mutants can be rapidly identified by color phenotype on indicator plates without extensive screening. This resolves the contradiction by achieving accurate disruption mutant identification through simple visual color changes, dramatically reducing screening time and resource consumption.
Solution Approach 2:
The patent enables disruption mutants to self-identify through phenotypic markers linked to the targeting cassette. The selectable and screening markers provide automatic visual identification of successful disruption events without requiring labor-intensive screening procedures. This self-service approach resolves the contradiction by allowing accurate identification of disruption mutants through inherent phenotypic markers, minimizing time and resource investment.
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
This method significantly increases the frequency of precise gene replacement and disruption events, overcoming the limitations of low homologous recombination efficiencies and random integration, facilitating efficient genetic modifications and strain engineering for biotechnological applications.
Implementation Method 1
the integrated targeting cassette is excised by a recombinase to leave behind one site-specific recombination site on the target locus
Implementation Method 2
the target locus in genome is replaced by homologous recombination with a targeting cassette
Data Source
AI summary
Provided are a novel two-step gene targeting method and a nucleotide construct for gene targeting. The method can improve the gene targeting efficiency and accurately identify a target gene knock-out mutant. The method of the present invention comprises: firstly, efficiently replacing a target gene in a genome with a targeting box by homologous recombination, the targeting box consisting of a target gene activity variant, a marker gene and site-specific recombination sites; and secondly, resecting the targeting box by recombinase, leaving a site-specific recombination site on the target gene to generate a target gene knock-out mutant, and removing a recombinase expression vector from the knock-out mutant by using a counter selection marker in the recombinase expression vector.