Bacterial Editing via Engine and Editing Vectors
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Solution Overview
Problem
Current methods for nucleic acid-guided nuclease editing in bacteria lack efficiency in increasing the percentage and diversity of edited cells, leading to a high background of unedited cells, which hinders the identification of precise genetic changes.
Innovation Solution
The method involves providing electrocompetent bacteria cells with engine and editing vectors that include promoters for nucleic acid-guided nucleases and recA proteins, allowing for multiple rounds of editing and selection to enhance editing rates, using automated multi-module cell processing instruments for efficient transformation and editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If nucleic acid-guided nuclease editing is performed in bacteria using conventional methods, then genetic edits can be introduced, but the percentage and diversity of edited cells remains low with high background of unedited cells
Solution Approach 1:
The patent applies preliminary action by pre-making bacteria cells electrocompetent before transformation, and by designing engine vectors with promoters that drive expression of nucleic acid-guided nucleases and recA proteins. This preparation in advance enables more efficient editing during the actual transformation process, increasing both the percentage of edited cells and the diversity of editing events while maintaining precision.
Solution Approach 2:
The patent employs parameter changes by optimizing transformation conditions for electrocompetent bacteria, adjusting promoter strength and composition in engine vectors, and modifying the ratio of engine vector to editing vector. These parameter adjustments significantly increase editing rates and the proportion of multiply-edited cells while preserving editing precision through controlled nuclease expression.
2Adaptability or versatility
If multiple editing cassettes are introduced to increase editing diversity, then more genomic locations can be edited simultaneously, but the complexity of the system increases
Solution Approach 1:
The patent applies segmentation by dividing the editing system into separate engine vectors and editing vectors. The engine vector contains the nuclease expression cassette and recA protein, while editing vectors contain individual or multiple editing cassettes with gRNA and donor DNA. This segmentation allows flexible combination of multiple editing cassettes to achieve diverse genomic edits without overwhelming system complexity, as each component has a dedicated function.
Solution Approach 2:
The patent implements universality through the engine vector design that can support multiple different editing vectors. The promoter system and recA protein expression in the engine vector work universally with various editing cassettes containing different gRNA and donor DNA combinations. This multi-functional design enables the same engine vector to facilitate diverse editing outcomes across multiple genomic locations simultaneously.
Data Source
AI summary
The present disclosure relates to methods for increasing observed editing rates in the surviving bacteria cells. The compositions and methods presented herein in combination lead to a phenomenon of “edit or die.” Although less cells survive plating and editing, a large percentage of cells that do survive are multiple editors. In one experiment it was found that if a cell survives transformation, plating, and editing, 75% of the surviving cells are multiple editors; that is, 75% of the surviving cells were simultaneously edited with edits at two or more different locations within the bacterial genome.