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Updated: Dec 5, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

A compact Cascade-Cas3 system for targeted genome engineering.

Bálint Csörgő^1,2,3, Lina M León^1,3, Ilea J Chau-Ly⁴

¹Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA.

|October 20, 2020

Summary

This summary is machine-generated.

This study introduces a novel CRISPR-Cas system using the Cas3 enzyme for large bacterial genome deletions, offering a powerful tool for genetic engineering and strain development.

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Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

Published on: January 3, 2015

Area of Science:

Molecular Biology
Genetics
Microbiology

Background:

CRISPR-Cas systems are powerful gene-editing tools.
Existing Cas9 and Cas12a enzymes are limited in creating large genomic deletions.

Purpose of the Study:

To develop a CRISPR-Cas system for efficient large DNA deletions in bacteria.
To engineer bacterial strains for synthetic biology and genome reduction.

Main Methods:

Utilized a Type I-C Cascade-Cas3 system for targeted genome engineering in bacteria.
Employed a single CRISPR RNA to guide Cas3 for DNA cleavage, creating large deletions (7-424 kb).
Exploited bidirectional deletion generation by Cas3 to reduce the Pseudomonas aeruginosa genome.

Main Results:

Achieved near-100% efficiency in generating large deletions in Pseudomonas aeruginosa.
Cas3 system outperformed Cas9, which produced only small deletions and point mutations.
Successfully reduced the P. aeruginosa genome by 13.5% (837 kb) using Cas3-mediated deletions.
Demonstrated efficient specification of deletion boundaries using homology-directed repair with Cascade-Cas3.
Showcased a transferable 'all-in-one' vector functional across multiple bacterial species (E. coli, P. syringae, K. pneumoniae).

Conclusions:

The Type I-C Cascade-Cas3 system (PaeCas3c) enables efficient and precise large genomic deletions in bacteria.
This technology has significant applications in synthetic biology, genome minimization, and targeted removal of genomic regions.
The developed system offers a versatile and efficient approach for bacterial strain manipulation.