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Updated: Jun 14, 2025
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
Published on: September 2, 2021
An improved CRISPR and CRISPR interference (CRISPRi) toolkit for engineering the model methanogenic archaeon
Qing Du1,2, Yufei Wei1,3, Liuyang Zhang1
1State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, No.1 Beichen West Road, Beijing, 100101, China.
Background:
The type II based CRISPR-Cas system remains restrictedly utilized in archaea, a featured domain of life that ranks parallelly with Bacteria and Eukaryotes. Methanococcus maripaludis, known for rapid growth and genetic tractability, serves as an exemplary model for studying archaeal biology and exploring CO2-based biotechnological applications. However, tools for controlled gene regulation remain deficient and CRISPR-Cas tools still need improved in this archaeon, limiting its application as an archaeal model cellular factory.
Results:
This study not only improved the CRISPR-Cas9 system for optimizing multiplex genome editing and CRISPR plasmid construction efficiencies but also pioneered an effective CRISPR interference (CRISPRi) system for controlled gene regulation in M. maripaludis. We developed two novel strategies for balanced expression of multiple sgRNAs, facilitating efficient multiplex genome editing. We also engineered a strain expressing Cas9 genomically, which simplified the CRISPR plasmid construction and facilitated more efficient genome modifications, including markerless and scarless gene knock-in. Importantly, we established a CRISPRi system using catalytic inactive dCas9, achieving up to 100-fold repression on target gene. Here, sgRNAs targeting near and downstream regions of the transcription start site and the 5'end ORF achieved the highest repression efficacy. Furthermore, we developed an inducible CRISPRi-dCas9 system based on TetR/tetO platform. This facilitated the inducible gene repression, especially for essential genes.
Conclusions:
Therefore, these advancements not only expand the toolkit for genetic manipulation but also bridge methodological gaps for controlled gene regulation, especially for essential genes, in M. maripaludis. The robust toolkit developed here paves the way for applying M. maripaludis as a vital model archaeal cell factory, facilitating fundamental biological studies and applied biotechnology development of archaea.
Insights
Researchers improved CRISPR-Cas9 tools for Methanococcus maripaludis, enabling efficient genome editing and gene regulation. This work enhances its use as an archaeal model for biotechnology and fundamental research.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- CRISPR-Cas systems are underutilized in archaea.
- Methanococcus maripaludis is a key model organism for archaeal biology and CO2-based biotechnology.
- Limited genetic tools hinder M. maripaludis applications.
Purpose of the Study:
- To enhance CRISPR-Cas9 tools for genome editing in M. maripaludis.
- To develop a CRISPR interference (CRISPRi) system for controlled gene regulation.
- To expand M. maripaludis as a model archaeal cell factory.
Main Methods:
- Developed strategies for balanced expression of multiple single-guide RNAs (sgRNAs).
- Engineered a Cas9-expressing strain for simplified plasmid construction and efficient gene knock-in.
- Established a CRISPR interference (CRISPRi) system using catalytically inactive dCas9 for gene repression.
- Created an inducible CRISPRi-dCas9 system using the TetR/tetO platform.
Main Results:
- Achieved efficient multiplex genome editing and CRISPR plasmid construction.
- Enabled markerless and scarless gene knock-in.
- Demonstrated up to 100-fold gene repression using CRISPRi.
- Successfully implemented inducible gene repression, particularly for essential genes.
Conclusions:
- Expanded the genetic manipulation toolkit for M. maripaludis.
- Bridged methodological gaps in controlled gene regulation, especially for essential genes.
- Paved the way for M. maripaludis as a vital archaeal cell factory for research and biotechnology.
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