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Related Experiment Video

Updated: Aug 6, 2025

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

Genome manipulation by guide-directed Argonaute cleavage.

Shan Huang¹, Kaihang Wang², Stephen L Mayo^1,2

¹Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 114-96, 1200 East California Boulevard, Pasadena, CA 91125, USA.

Nucleic Acids Research

|March 17, 2023

Summary

This summary is machine-generated.

Prokaryotic Argonautes (pAgos) can be mutagenic, inducing DNA cleavage and recombination in bacterial genomes. This guide-directed DNA interference has potential applications in genetic engineering and pAgo characterization.

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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

Area of Science:

Molecular Biology
Genetics
Microbiology

Background:

Prokaryotic Argonautes (pAgos) are known to mediate DNA interference using guide DNA to cleave target DNA.
Clostridium butyricum Ago (CbAgo) induces DNA interference and double-stranded breaks (DSBs) in target DNA, aiding host defense against foreign DNA.
The mutagenic potential of CbAgo-mediated DNA cleavage remained uninvestigated.

Purpose of the Study:

To investigate whether CbAgo-mediated DNA cleavage is mutagenic.
To explore the mechanisms underlying CbAgo-induced DNA cleavage and recombination in Escherichia coli.
To assess the utility of CbAgo's mutagenic activity in genetic manipulation.

Main Methods:

Utilized plasmid-encoded guide sequences to direct CbAgo cleavage of target sites in the E. coli genome.
Analyzed chromosome recombination between homologous sequences induced by CbAgo cleavage.
Studied the role of DSBs and RecBCD processing in the recombination mechanism.
Investigated the impact of CbAgo cleavage on cell growth in RecA-deficient E. coli strains.

Main Results:

CbAgo-directed cleavage of genomic target sites induced chromosome recombination between homologous sequences in E. coli.
Recombination rates correlated with CbAgo DNA cleavage activity.
Mechanistic studies indicated that DSBs and RecBCD processing are involved in the recombination process.
Guide-directed CbAgo cleavage on chromosomes severely impaired cell growth in RecA-deficient E. coli, enabling counter-selection for Lambda-Red recombineering.

Conclusions:

Guide-directed CbAgo cleavage of the host genome is mutagenic, leading to outcomes dependent on host DNA repair pathways.
This DNA-guided interference mechanism offers potential for broader genetic manipulation applications.
The study provides an in vivo assay for characterizing and engineering pAgo DNA cleavage activity.