Application of Cas12j for Streptomyces Editing

Author:

Tan Lee Ling1ORCID,Heng Elena1,Leong Chung Yan2ORCID,Ng Veronica2,Yang Lay Kien2ORCID,Seow Deborah Chwee San2,Koduru Lokanand1,Kanagasundaram Yoganathan2ORCID,Ng Siew Bee2,Peh Guangrong3ORCID,Lim Yee Hwee3ORCID,Wong Fong Tian13ORCID

Affiliation:

1. Institute of Molecular and Cell Biology (IMCB), Agency for Science, Technology and Research (A*STAR), 61 Biopolis Drive, Proteos #07-06, Singapore 138673, Singapore

2. Singapore Institute of Food and Biotechnology Innovation (SIFBI), Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way, Nanos #02-01, Singapore 138669, Singapore

3. Institute of Sustainability for Chemicals, Energy and Environment (ISCE2), Agency for Science, Technology and Research (A*STAR), 8 Biomedical Grove, Neuros #07-01, Singapore 138665, Singapore

Abstract

In recent years, CRISPR-Cas toolboxes for Streptomyces editing have rapidly accelerated natural product discovery and engineering. However, Cas efficiencies are oftentimes strain-dependent, and the commonly used Streptococcus pyogenes Cas9 (SpCas9) is notorious for having high levels of off-target toxicity effects. Thus, a variety of Cas proteins is required for greater flexibility of genetic manipulation within a wider range of Streptomyces strains. This study explored the first use of Acidaminococcus sp. Cas12j, a hypercompact Cas12 subfamily, for genome editing in Streptomyces and its potential in activating silent biosynthetic gene clusters (BGCs) to enhance natural product synthesis. While the editing efficiencies of Cas12j were not as high as previously reported efficiencies of Cas12a and Cas9, Cas12j exhibited higher transformation efficiencies compared to SpCas9. Furthermore, Cas12j demonstrated significantly improved editing efficiencies compared to Cas12a in activating BGCs in Streptomyces sp. A34053, a strain wherein both SpCas9 and Cas12a faced limitations in accessing the genome. Overall, this study expanded the repertoire of Cas proteins for genome editing in actinomycetes and highlighted not only the potential of recently characterized Cas12j in Streptomyces but also the importance of having an extensive genetic toolbox for improving the editing success of these beneficial microbes.

Funder

Singapore National Research Foundation (NRF)’s 19th Competitive Research Program

Agency for Science, Technology and Research (A*STAR), Singapore

Publisher

MDPI AG

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