One-Vector Genome-Modifying Enzyme Screening With Integrated Target Sites
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Solution Overview
Problem
Existing systems for identifying genome modifying enzymes, such as recombinases and CRISPR/Cas nucleases, are inefficient for high-throughput screening of large enzyme libraries due to the need for co-transfection of two vectors, which complicates the process and introduces transfection variations.
Innovation Solution
An integrated one-vector system that combines the enzyme polypeptide and its specific target site sequence within a single vector, along with a unique identifier, facilitating high-throughput screening and minimizing transfection variations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a two-vector system is used to express candidate genome modifying enzymes and target site sequences, then enzyme identification can be performed, but the system complexity increases and transfection variations are introduced
Solution Approach 1:
The patent combines the candidate genome modifying enzyme coding sequence and the target site sequence into a single integrated vector. This merging eliminates the need for co-transfection of two separate vectors, thereby reducing system complexity and minimizing transfection variations while maintaining the ability to identify active enzymes through intracellular recombination events
2Reliability
If a two-vector system is used for enzyme screening, then enzyme function can be assessed, but the screening process becomes less efficient for high-throughput applications
Solution Approach 1:
By integrating both the enzyme coding sequence and target site into one vector, the patent enables single-transfection protocols that are significantly more efficient for high-throughput screening. This approach allows hundreds to thousands of candidate enzymes to be screened in parallel without the logistical complexities of managing two separate vector transfections
Solution Approach 2:
The integrated vector design enables the system to self-assess enzyme function through autonomous intracellular recombination events. When the expressed enzyme successfully modifies the target site within the same cell, it creates a detectable recombination event that automatically reports enzyme activity, eliminating the need for complex external assessment protocols
Data Source
AI summary
The present application relates to an integrated one vector system for identifying a genome modifying enzyme, e.g., from a large library. The one-vector system integrates the components for enzyme identification into a single vector to facilitate a high throughput screening process.