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Updated: Jul 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

Bacterial genome engineering using CRISPR-associated transposases.

Diego Rivera Gelsinger^1,2, Phuc Leo H Vo^3,4, Sanne E Klompe^1,5

¹Department of Biochemistry and Molecular Biophysics, Columbia University, New York, NY, USA.

Nature Protocols

|January 12, 2024

Summary

This summary is machine-generated.

CRISPR-associated transposases enable efficient, kilobase-scale bacterial genome engineering without homologous recombination. This protocol details using CRISPR-associated transposase (CAST) systems for precise genetic payload integration in diverse bacteria.

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Area of Science:

Molecular Biology
Genomics
Synthetic Biology

Background:

CRISPR-associated transposases (CAST) offer a novel platform for large-scale genome engineering.
Existing methods often require homologous recombination and struggle with large genetic payloads.
CAST systems provide accurate, programmable integration of DNA sequences.

Purpose of the Study:

To present a detailed protocol for bacterial genome engineering using CAST systems.
To provide guidelines for vector selection, guide RNA and DNA payload customization, and delivery methods.
To introduce computational tools for guide RNA design and multiplexed DNA insertion.

Main Methods:

Utilized CRISPR RNA-guided transposases for genomic insertions in Escherichia coli.
Developed a computational algorithm for designing guide RNAs to minimize off-target effects.
Implemented a CRISPR array cloning pipeline for multiplexed DNA insertions.

Main Results:

Achieved near 100% efficiency for genomic insertions in E. coli.
Demonstrated robust functionality of CAST systems in diverse Gram-negative bacteria.
Enabled multiplexed edits through programming with multiple guides.
Facilitated isolation of clonal strains with novel genomic integrations within 1-2 weeks.

Conclusions:

CRISPR-associated transposase systems represent a powerful tool for kilobase-scale genome engineering.
The presented protocol simplifies and enhances the efficiency of bacterial genome modification.
This technology has broad applicability in engineering diverse bacterial species.