An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae

Bo-Chou Chen; Yu-Zhen Chen; Huan-Yu Lin

doi:10.3390/biom13101561

An Introduced RNA-Only Approach for Plasmid Curing via the CRISPR-Cpf1 System in Saccharomyces cerevisiae

Biomolecules. 2023 Oct 23;13(10):1561. doi: 10.3390/biom13101561.

Authors

Bo-Chou Chen¹, Yu-Zhen Chen², Huan-Yu Lin¹

Affiliations

¹ Bioresource Collection and Research Center, Food Industry Research and Development Institute, Hsinchu 300, Taiwan.
² Department of Food Science and Technology, Hungkuang University, Taichung 433, Taiwan.

Abstract

The CRISPR-Cas system has been widely used for genome editing due to its convenience, simplicity and flexibility. Using a plasmid-carrying Cas protein and crRNA or sgRNA expression cassettes is an efficient strategy in the CRISPR-Cas genome editing system. However, the plasmid remains in the cells after genome editing. Development of general plasmid-curing strategies is necessary. Based on our previous CRISPR-Cpf1 genome-editing system in Saccharomyces cerevisiae, the crRNA, designed for the replication origin of the CRISPR-Cpf1 plasmid, and the ssDNA, as a template for homologous recombination, were introduced for plasmid curing. The efficiency of the plasmid curing was 96 ± 4%. In addition, we further simplified the plasmid curing system by transforming only one crRNA into S. cerevisiae, and the curing efficiency was about 70%. In summary, we have developed a CRISPR-mediated plasmid-curing system. The RNA-only plasmid curing system is fast and easy. This plasmid curing strategy can be applied in broad hosts by designing crRNA specific for the replication origin of the plasmid. The plasmid curing system via CRISPR-Cas editing technology can be applied to produce traceless products without foreign genes and to perform iterative processes in multiple rounds of genome editing.

Keywords: CRISPR-Cpf1; Saccharomyces cerevisiae; genome-editing; plasmid-curing.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Gene Editing* / methods
Plasmids / genetics
RNA / metabolism
RNA, Guide, CRISPR-Cas Systems
Saccharomyces cerevisiae* / genetics
Saccharomyces cerevisiae* / metabolism

Substances

RNA
RNA, Guide, CRISPR-Cas Systems

Grants and funding

This research was funded by the Ministry of Economic Affairs, Taiwan, grant number 111-EC-17-A-22-0525 and 112-EC-17-A-22-0525. It was also funded by the National Science and Technology Council, grant number MOST 111-2635-B-080-001.