Protected DNA Templates for Scalable Homologous Recombination
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Solution Overview
Problem
Current genome editing technologies, such as HR, are labor-intensive, difficult to scale, and inefficient in organisms where HR is not prevalent, necessitating the development of more robust and scalable methods for genome engineering.
Innovation Solution
The use of a guide polynucleotide, a protected polynucleotide modification template, and a Cas endonuclease to modify nucleotide sequences and increase the frequency of homologous directed repair, while decreasing off-site integration of the modification template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional homologous recombination (HR) is used for genome editing, then gene modification can be achieved, but the process is labor-intensive and difficult to scale
Solution Approach 1:
The patent segments the genome editing process into distinct functional components: guide RNA for target recognition, Cas endonuclease for cleavage, and protected DNA templates for repair. This segmentation enables modular optimization and simplifies the overall process while maintaining high efficiency
Solution Approach 2:
The patent introduces protected DNA templates as intermediary molecules that mediate between the Cas endonuclease cleavage event and the final genome repair. These templates are designed with specific protective features that enable them to function as controlled intermediaries in the HR process, increasing efficiency while reducing procedural complexity
2Reliability
If traditional HR methods are used in organisms where HR is not efficient, then gene modification is difficult to achieve, but increasing HR efficiency may lead to off-site integration of modification templates
Solution Approach 1:
The patent changes key parameters of the DNA template molecule itself by adding protective modifications (such as phosphorothioate bonds or hairpin structures) at the ends of the template. This parameter change enables the template to withstand cellular conditions that normally promote off-site integration, while simultaneously enhancing its ability to participate in HR at the intended target site
Solution Approach 2:
The patent converts the potential harm of cellular mechanisms that cause off-site integration into a benefit by designing templates with protective features. These features inadvertently protect against off-site integration while promoting on-target HR, effectively turning a harmful cellular process into a selective advantage for properly designed templates
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 enhances the robustness of HR, making it more efficient and scalable, and reduces off-site integration, thereby improving the precision and effectiveness of genome editing.
Implementation Method 1
increase the frequency of homologous directed repair
Data Source
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AI summary
Compositions and methods are provided for modifying a nucleotide sequence in the genome of a cell. The methods and compositions employ a guide polynucleotide, a protected polynucleotide modification template and a Cas endonuclease to modify a nucleotide sequence and/or to increase the frequency of homologous directed repair. The methods can further be used to decrease the frequency of off-site integration of any modification template. The present disclosure also describes methods for selecting a cell comprising a modified target site in its genome and methods for selecting a cell comprising a polynucleotide of interest inserted into a target site in its genome.