CRISPR-Cas12a-Assisted Genome Editing in Amycolatopsis mediterranei

Yajuan Zhou,Guoping Zhao,Jiacheng Wu,Xinqiang Liu,Jin Wang

doi:10.3389/fbioe.2020.00698

Abstract

Amycolatopsis mediterranei U32 is an industrial producer of rifamycin SV, whose derivatives have long been the first-line antimycobacterial drugs. In order to perform genetic modification in this important industrial strain, a lot of efforts have been made in the past decades and a homologous recombination-based method was successfully developed in our laboratory, which, however, requires the employment of an antibiotic resistance gene for positive selection and did not support convenient markerless gene deletion. Here in this study, the clustered regularly interspaced short palindromic repeat (CRISPR) system was employed to establish a genome editing system in A. mediterranei U32. Specifically, the Francisella tularensis subsp. novicida Cas12a (FnCas12a) gene was first integrated into the U32 genome to generate target-specific double-stranded DNA (dsDNA) breaks (DSBs) under the guidance of CRISPR RNAs (crRNAs). Then, the DSBs could be repaired by either the non-homologous DNA end-joining (NHEJ) system or the homology-directed repair (HDR) pathway, generating inaccurate or accurate mutations in target genes, respectively. Besides of A. mediterranei, the present work may also shed light on the development of CRISPR-assisted genome editing systems in other species of the Amycolatopsis genus.

Highlights

Amycolatopsis mediterranei U32 is an industrial strain for production of rifamycin SV (Zhao et al, 2010), the first-line drug for anti-mycobacterial therapy till (Rothstein, 2016)
The Amycolatopsis genus is well known to produce a huge diversity of secondary metabolites (Xu et al, 2014; Kumari et al, 2016; Adamek et al, 2018), the lack of efficient genome editing technology has severely impeded the research progress in this genus
To test the effectiveness of Cas12a-mediated site-specific DSBs, Cas12a and CRISPR RNAs (crRNAs) were co-expressed in U32

Summary

Introduction

Amycolatopsis mediterranei U32 is an industrial strain for production of rifamycin SV (Zhao et al, 2010), the first-line drug for anti-mycobacterial therapy till (Rothstein, 2016). To facilitate the study of rifamycin biosynthesis as well as the molecular bioengineering of the producer, a genetic manipulation method based on native homologous recombination was developed for gene knockout in A. mediterranei (Ding et al, 2003). Due to the relatively low efficiency of both DNA transformation and homologous recombination in A. mediterranei, an antibiotic cassette is usually employed to replace the target gene and the transformants are grown under antibiotic selection. Due to the CRISPR-Cas12a-Assisted Genome Editing relatively low genetic engineering efficiency, there are very few reports of successful construction of a markerless mutant in A. mediterranei. The Amycolatopsis genus is well known to produce a huge diversity of secondary metabolites (Xu et al, 2014; Kumari et al, 2016; Adamek et al, 2018), the lack of efficient genome editing technology has severely impeded the research progress in this genus

Methods

Results

Conclusion