Genomic Integration via Targeted Double-Strand Breaks
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Solution Overview
Problem
Current genetic engineering techniques are inefficient for simultaneous integration of multiple exogenous nucleic acids into specific regions of a host cell genome, requiring serial engineering cycles and reliance on selectable markers, which limits the rapid construction of complex biosynthetic pathways in industrial microbes.
Innovation Solution
The method involves introducing multiple exogenous nucleic acids and site-specific nucleases into a host cell to induce targeted double-strand breaks, leveraging the native homologous recombination machinery for simultaneous integration at multiple loci without the need for selectable markers, using homology regions and nucleases that recognize specific target sites within the genome.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If traditional gene targeting methods are used to integrate exogenous nucleic acids, then integration can occur at specific genomic loci, but the efficiency is extremely low (only ~1 in 10^6 events)
Solution Approach 1:
The patent introduces designer nucleases (such as zinc finger nucleases, TALENs, or CRISPR-Cas systems) that create targeted double-strand breaks at the desired integration sites before the actual integration event. This preliminary action of creating controlled breaks dramatically enhances the efficiency of homologous recombination, allowing integration frequencies to increase from ~10^-6 to >10^-2 events per transformation
2Adaptability or versatility
If multiple rounds of serial engineering are performed to integrate multiple DNA assemblies, then complex biosynthetic pathways can be constructed, but the process requires many sequential steps and extensive time
Solution Approach 1:
The patent enables simultaneous integration of multiple exogenous nucleic acid assemblies (containing different genes or pathway components) into the host genome in a single transformation event. By using multiple designer nucleases targeting different genomic loci and providing corresponding donor DNA assemblies, the system achieves multi-locus integration in parallel, reducing the number of engineering cycles from multiple sequential steps to a single step
3Difficulty of detecting and measuring
If selectable markers are used to identify successful integration events, then integration can be detected, but the process becomes more complex and requires additional genetic elements
Solution Approach 1:
The patent employs transient expression of designer nucleases from plasmids or viral vectors that do not integrate into the host genome. The nucleases perform their function of creating double-strand breaks and then are degraded, leaving no permanent trace. This disposable approach eliminates the need for selectable markers or other permanent genetic elements, simplifying the final engineered organism
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 approach significantly increases the frequency of successful integration of multiple nucleic acids into specific genomic locations, reducing the number of engineering cycles required and enabling the rapid construction of complex metabolic pathways, such as the isoprenoid pathway, with high efficiency and markerless recovery.
Implementation Method 1
inducing targeted double-strand breaks in the host cell's genome at the intended sites of integration
Implementation Method 2
The methods and compositions utilize the native homologous recombination machinery of the host cell
Data Source
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AI summary
Provided herein are methods of integrating one or more exogenous nucleic acids into one or more selected target sites of a host cell genome. In certain embodiments, the methods comprise contacting the host cell genome with one or more integration polynucleotides comprising an exogenous nucleic acid to be integrated into a genomic target site, and a nuclease capable of causing a double-strand break near or within the genomic target site.