Excisable Nucleic Acid Construct for Precise Genomic Excision
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Solution Overview
Problem
Current genetic engineering methods for removing target nucleic acids from host cells are limited by low frequency and high frequency of genomic instabilities, such as deleterious recombinase binding sites and low excision events, necessitating the development of methods for precise and efficient excision without instability.
Innovation Solution
The use of an excisable nucleic acid construct comprising a tandem repeat and homing endonuclease recognition sites allows for high frequency and high fidelity excision of target nucleic acids from host cells, facilitated by homing endonucleases like I-SceI or F-CphI, which cleave the construct and enable intrachromosomal recombination for precise repair.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If site specific recombinase methods are used for removal of target nucleic acids, then excision can be achieved, but deleterious recombinase binding sites are left behind creating potential genomic instabilities
Solution Approach 1:
The patent extracts and removes the harmful recombinase binding sites from the excision mechanism by using homing endonucleases instead of recombinases. The homing endonuclease recognizes and cleaves specific DNA sequences, and after excision, the binding sites are completely removed rather than left behind in the genome, thus eliminating the source of genomic instability.
Solution Approach 2:
The patent changes the molecular mechanism parameter from recombinase-based site-specific recombination to homing endonuclease-based cleavage and repair. This parameter change transforms the excision process into one that does not leave behind harmful binding sites, thereby improving genomic stability while maintaining excision capability.
2Productivity
If traditional excision methods are used, then target nucleic acid removal can occur, but excision events happen at low frequency necessitating growth-selection of rare host cells
Solution Approach 1:
The patent implements a self-service mechanism where the homing endonuclease automatically recognizes and cleaves the target nucleic acid sequence with high efficiency. The system includes built-in selection markers that automatically identify and enable growth of successfully excised cells, eliminating the need for time-consuming manual selection processes and significantly increasing excision frequency.
3Adaptability or versatility
If multiple genetic engineering events are performed, then genetic modification capability is enhanced, but accumulation of harmful residues and genomic instabilities increases
Solution Approach 1:
The patent extracts harmful residues from the genetic engineering process by using homing endonuclease-based excision that completely removes target sequences including any potential harmful elements. Each excision event leaves a clean scar-free genome, enabling multiple sequential genetic engineering events without cumulative harmful effects or genomic instability.
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 enables precise and efficient removal of target nucleic acids, reducing genomic instability and allowing for 'marker recycling' and regulation of gene expression, while enabling multiple genetic engineering events without leaving harmful residues.
Implementation Method 1
A homing endonuclease bound to a homing endonuclease recognition site can cleave the excisable nucleic acid construct at or adjacent to the homing endonuclease recognition site
Implementation Method 2
The first and second tandem repeat nucleic acids independently comprise at least 18 nucleotide base pairs... repair of the host cell genome can occur through intrachromosomal recombination facilitated by the first and second tandem repeats
Data Source
AI summary
Nucleic acids, compositions, and methods that allow for the excision of one or more loci from the genome of a host cell are provided herein. In particular, provided herein is an excisable nucleic acid construct comprising, in a 5′ to 3′ orientation: a first tandem repeat nucleic acid, a first homing endonuclease recognition site, a target nucleic acid, a second homing endonuclease recognition site, and a second tandem repeat nucleic acid. In some embodiments, the excisable nucleic acid construct is integrated into the host cell genome, and the target nucleic acid can be excised from the host cell genome by contacting the homing endonuclease recognition sites with one or more appropriate homing endonucleases.