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CRISPR/Cas9 Genome Editing

CRISPR/Cas9 Genome Editing

The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...

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Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

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CRISPR-dCas12a-mediated genetic circuit cascades for multiplexed pathway optimization.

Yaokang Wu^1,2, Yang Li^1,2, Ke Jin^1,2

¹Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi, China.

Nature Chemical Biology

|January 16, 2023

Summary

This study introduces a

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Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

Published on: June 2, 2018

Area of Science:

Synthetic Biology
Metabolic Engineering
Microbial Biotechnology

Background:

Microbial cell factory efficiency is often limited by challenges in coordinating multiple genetic targets.
Optimizing complex metabolic pathways requires advanced regulatory tools.

Purpose of the Study:

To develop and apply a 'design-build-test-learn' framework for efficient, coordinated optimization of metabolic pathways.
To engineer Bacillus subtilis for enhanced production of glucosamine-6-phosphate (GlcN6P) and its derivatives.

Main Methods:

A platform strain of Bacillus subtilis was constructed with biosensor signal-amplifying and genetic regulation circuits.
A synthetic CRISPR RNA array blend for boosting and leading (ScrABBLE) device was employed to generate 5,184 combinatorial gene assemblies targeting three genes.
High-throughput screening and metabolic engineering were used to identify and optimize the best producers.

Main Results:

The ScrABBLE device enabled efficient multiplexed optimization of the GlcN6P biosynthesis pathway.
Engineered strains achieved high titers of valuable pharmaceuticals N-acetylglucosamine and N-acetylmannosamine.
A N-acetylglucosamine titer of 183.9 g/liter was reached in a 15-liter bioreactor.

Conclusions:

The 'design-build-test-learn' framework and ScrABBLE device offer a powerful strategy for microbial cell factory optimization.
This approach facilitates the efficient production of high-value compounds from microbial systems.
The ScrABBLE device demonstrates potential for broad, generic applications in synthetic biology.