Multiplex Automated Genome Engineering via Cyclic DNA Addition
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Solution Overview
Problem
Current genome engineering methods are inefficient, typically introducing only one DNA construct per cell at low efficiency, requiring multiple steps and selection processes to eliminate unwanted cells, making it rare to complete a genome engineering construct with more than a dozen steps.
Innovation Solution
A method for introducing multiple nucleic acid sequences into cells using an automated system involving transformation or transfection with a nucleic acid oligomer, followed by incubation in growth medium, repeated until multiple sequences are introduced, allowing for cyclic addition of DNAs in parallel with or without selectable markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current methods introduce one DNA construct per cell, then the process is simple, but the efficiency is low (around 0.1%) and requires many steps
Solution Approach 1:
The patent segments the DNA construct introduction process into multiple parallel cycles, where each cycle introduces one or more DNA constructs to multiple cells simultaneously. This segmentation allows the complex task of introducing multiple constructs to be broken down into manageable, repeatable units that can be performed in parallel, thereby increasing efficiency while controlling complexity
Solution Approach 2:
The patent employs periodic cyclic addition of DNA constructs to cells. The process repeats cycles of DNA addition, incubation, and selection, where each cycle builds upon the previous one. This periodic action enables systematic introduction of multiple constructs through repeated, manageable iterations rather than attempting to introduce all constructs in a single complex step
2Device complexity
If multiple DNA constructs are introduced into cells simultaneously, then the number of steps is reduced, but the complexity of the process increases
Solution Approach 1:
The patent segments the simultaneous introduction of multiple DNA constructs into a series of simpler, cyclic steps. Each cycle involves introducing one or more DNA constructs, followed by incubation and selection. This segmentation transforms a complex simultaneous introduction process into multiple simpler, manageable steps that can be systematically performed
Solution Approach 2:
The patent employs preliminary selection steps between DNA introduction cycles to ensure that cells have properly incorporated the desired constructs before the next cycle begins. This preliminary action simplifies the overall process by pre-screening cells and eliminating those that have not properly integrated the DNA, thereby reducing the complexity of tracking and managing multiple constructs
3Reliability
If selection and screen processes are performed at each step, then unwanted cells are eliminated, but the time required increases
Solution Approach 1:
The patent incorporates selection steps within the periodic cycles of DNA introduction. Each cycle of DNA addition is followed by incubation and selection, creating a rhythmic pattern where selection occurs at regular intervals. This periodic selection efficiently eliminates unwanted cells at the appropriate stages without requiring continuous or excessive selection steps, thereby reducing overall time while maintaining reliability
Solution Approach 2:
The patent maintains continuous progress by overlapping DNA introduction, incubation, and selection steps. Rather than completing all DNA introduction steps before beginning selection, the process continuously cycles through these steps, ensuring that useful actions (DNA integration and selection) occur without unnecessary delays. This continuity reduces total time while maintaining the reliability of eliminating unwanted cells
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 enables efficient introduction of multiple nucleic acid sequences into cells, facilitating combinatorial exploration and genome engineering, increasing the efficiency of allele replacement and reducing the number of steps required for genome construction.
Implementation Method 1
transforming or transfecting the cell using transformation medium or transfection medium including at least one nucleic acid oligomer
Implementation Method 2
transforming or transfecting the cell using transformation medium or transfection medium including at least one nucleic acid oligomer
Data Source
AI summary
The present invention relates to automated methods of introducing multiple nucleic acid sequences into one or more target cells.