CRISPR/Cas13d-Mediated Microbial RNA Knockdown

Kun Zhang; Zhihui Zhang; Jianan Kang; Jiuzhou Chen; Jiao Liu; Ning Gao; Liwen Fan; Ping Zheng; Yu Wang; Jibin Sun

doi:10.3389/fbioe.2020.00856

CRISPR/Cas13d-Mediated Microbial RNA Knockdown

Front Bioeng Biotechnol. 2020 Jul 30:8:856. doi: 10.3389/fbioe.2020.00856. eCollection 2020.

Authors

Kun Zhang^{1

2}, Zhihui Zhang^{1

2}, Jianan Kang³, Jiuzhou Chen¹, Jiao Liu¹, Ning Gao^{1

2}, Liwen Fan^{1

4}, Ping Zheng^{1

2

4}, Yu Wang^{1

2}, Jibin Sun^{1

2}

Affiliations

¹ Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China.
² University of Chinese Academy of Sciences, Beijing, China.
³ College of Life Engineering, Shenyang Institute of Technology, Fushun, China.
⁴ School of Life Sciences, University of Science and Technology of China, Hefei, China.

Abstract

RNA-guided and RNA-targeting type IV-D CRISPR/Cas systems (CRISPR/Cas13d) have recently been identified and employed for efficient and specific RNA knockdown in mammalian and plant cells. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence (PFS)-independent manner. These properties make this system a promising tool for multiplex gene expression regulation in microbes. Herein, we aimed to establish a CRISPR/Cas13d-mediated RNA knockdown platform for bacterial chassis. CasRx, Cas13d from Ruminococcus flavefaciens XPD3002, was selected due to its high activity. However, CasRx was found to be highly toxic to both Escherichia coli and Corynebacterium glutamicum, especially when it cooperated with its guide and target RNAs. After employing a low copy number vector, a tightly controlled promoter, and a weakened ribosome binding site, we successfully constructed an inducible expression system for CasRx and applied it for repressing the expression of a green fluorescent protein (GFP) in E. coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30-50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.

Keywords: CRISPR; Cas13d; CasRx; RNA knockdown; type IV-D CRISPR effector.