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Updated: Jul 31, 2025
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Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
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Efficient Genome and Base Editing in Human Cells Using ThermoCas9.
Despoina Trasanidou1,2, Patrick Barendse1,2, Evgenios Bouzetos1,2
1Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands.
The CRISPR Journal
|May 3, 2023
Summary
A new CRISPR-Cas9 tool, ThermoCas9, from *Geobacillus thermodenitrificans* offers enhanced genome editing in human cells. It enables efficient gene disruption and a novel base editor, ThermoBE4, for precise C-to-T conversions.
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas9 gene editing predominantly uses Streptococcus pyogenes Cas9 (SpyCas9), which has limitations in genome targeting scope.
- Exploring alternative Cas9 orthologs is crucial for expanding genome engineering capabilities.
Purpose of the Study:
- To evaluate the activity and utility of a thermostable type II-C Cas9 ortholog from Geobacillus thermodenitrificans (ThermoCas9) in human cells.
- To develop and characterize a ThermoCas9-based base editor (ThermoBE4) for efficient C-to-T conversions.
Main Methods:
- Expression and functional validation of ThermoCas9 in human cell lines.
- Development of a ThermoCas9-mediated base editor (ThermoBE4).
- Comparative analysis of ThermoBE4 activity window against SpyCas9 base editor (BE4).
Main Results:
- ThermoCas9 is active in human cells and functions as an efficient genome editing tool, particularly for gene disruption.
- ThermoBE4 enables programmable nicking and C-to-T base editing.
- ThermoBE4 demonstrates a three-fold larger activity window compared to the SpyCas9-based BE4 editor.
Conclusions:
- ThermoCas9 serves as a valuable alternative to SpyCas9, expanding the targeting range for genome editing applications.
- ThermoCas9 offers a versatile platform for both standard genome editing and advanced base editing in human cells.
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