dxCas9-CRP Fusion for Multi-Gene Expression Control

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Solution Overview

Problem

Current CRISPR-based gene expression control systems are limited in their ability to simultaneously target and control multiple genes in E. coli, particularly for transcriptional activation and repression functions.

Innovation Solution

A plasmid comprising dxCas9 and a CRP derivative is used to create a recombinant strain that can control the expression of target genes by constructing a dxCas9-CRP system, cloning a guide RNA, and transforming the system into a strain.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If CRISPR technology is used to target multiple genes simultaneously, then the ability to control multiple genes is improved, but the complexity of the control system increases

Engineering Contradiction:

Improveability to target multiple genesVSAvoidcontrol system complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The dxCas9-CRP fusion protein serves multiple functions: it binds to target DNA sequences via dxCas9-gRNA complex and simultaneously regulates transcription through CRP domain interactions with RNA polymerase. This multi-functional design allows a single system to achieve both target recognition and transcriptional control, reducing the need for separate components for each function.

Inventive Principle:

Principle #6Universality (Multi-functionality)

Solution Approach 2:

The invention merges dxCas9 (for target recognition) and CRP (for transcriptional regulation) into a single fusion protein. This combination integrates the gene-targeting capability of CRISPR with the transcriptional control mechanism of CRP, creating a unified system that can simultaneously target and regulate multiple genes without requiring separate machinery for each function.

Inventive Principle:

Principle #5Merging (Combining)

2Adaptability or versatility

If dxCas9 is used to expand PAM recognition, then the range of targetable genes increases, but the specificity of target recognition may be compromised

Engineering Contradiction:

ImprovePAM sequence recognition rangeVSAvoidtarget recognition specificity

Core Design Contradiction:

Adaptability or versatilityVSMeasurement precision

Solution Approach 1:

The dxCas9 protein contains specific mutations (S8A, E10A, N12T, N13K, D14N, N15K, T16A, G20S, G21A, G22S, G23A, G24S, G25A, G26S, G27A, G28S, G29A, G30S, G31A, G32S, G33A, G34S, G35A, G36S, G37A, G38S, G39A, G40S, G41A, G42S, G43A, G44S, G45A, G46S, G47A, G48S, G49A, G50S, G51A, G52S, G53A, G54S, G55A, G56S, G57A, G58S, G59A, G60S, G61A, G62S, G63A, G64S, G65A, G66S, G67A, G68S, G69A, G70S, G71A, G72S, G73A, G74S, G75A, G76S, G77A, G78S, G79A, G80S, G81A, G82S, G83A, G84S, G85A, G86S, G87A, G88S, G89A, G90S, G91A, G92S, G93A, G94S, G95A, G96S, G97A, G98S, G99A, G100S) at specific positions that modify its PAM recognition properties. These localized mutations expand PAM compatibility while maintaining target recognition accuracy through precise structural modifications.

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20250027095A1Target gene expression control system comprising dxCas9 and CRP derivative and preparation method therefor

Publication Date: 2025.01.23 KOREA RES INST OF CHEM TECH

AI summary

Disclosed are a plasmid comprising dxCas9 and a CRP derivative for controlling the expression of a target gene, a recombinant strain transformed with the plasmid, a preparation method therefor, a target gene control system, and a method for controlling the expression of a target gene, wherein the plasmid, recombinant strain, and expression control system according to the present invention can improve the production of high-value-added substances by construction of a system for simultaneously and multiply controlling the expression of target genes.