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A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

Programmable type III-A CRISPR-Cas DNA targeting modules.

H Travis Ichikawa¹, John C Cooper¹, Leja Lo¹

¹Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia, United States of America.

|April 26, 2017

Summary

This summary is machine-generated.

Researchers engineered CRISPR-Cas systems for programmable DNA targeting. These Csm modules, expressed in E. coli, specifically eliminate invading plasmids activated by transcription, offering a new platform for genetic engineering.

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Area of Science:

Molecular Biology
Microbiology
Biotechnology

Background:

CRISPR-Cas systems are prokaryotic defense mechanisms against invaders.
Type III-A Csm CRISPR-Cas systems are widespread and target DNA/RNA.

Purpose of the Study:

To construct and express functional Csm modules from different bacteria in E. coli.
To investigate the transcription-dependent DNA targeting mechanism of Type III-A CRISPR-Cas systems.
To establish a programmable DNA targeting platform.

Main Methods:

Construction of Csm system modules (Cas6, CRISPR locus, Csm effector proteins) from L. lactis, S. epidermidis, and S. thermophilus.
Heterologous expression of functional modules in E. coli.
Assessment of plasmid elimination activity based on crRNA targeting and target sequence transcription.

Main Results:

Expressed Csm modules successfully eliminated invading plasmids recognized by crRNAs.
Plasmid targeting was dependent on transcription of the crRNA target sequence.
Activity was abolished when target transcription was blocked or non-target sequences were transcribed.
Csm modules could be reprogrammed for novel targets by altering crRNA coding sequences.

Conclusions:

Functional Type III-A Csm systems can be reconstituted and expressed in E. coli.
Transcription-activated DNA targeting is a key characteristic of these Csm systems.
These engineered systems offer a versatile platform for studying CRISPR-Cas mechanisms and developing programmable DNA targeting tools.