Abstract
Streptomyces strains produce a great number of valuable natural products. With the development of genome sequencing, a vast number of biosynthetic gene clusters with high potential for use in the discovery of valuable clinical drugs have been revealed. Therefore, emerging needs for tools to manipulate these biosynthetic pathways are presented. Although the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas 9) system has exhibited great capabilities for gene editing in multiple Streptomyces strains, it has failed to work in some newly discovered strains and some important industrial strains. Additionally, the protospacer adjacent motif (PAM) recognition scope of this system sometimes limits its applications for generating precise site mutations and insertions. Here, we developed three efficient CRISPR-FnCas12a systems for multiplex genome editing in several Streptomyces strains. Each system exhibited advantages for different applications. The CRISPR-FnCas12a1 system was efficiently applied in the industrial strain Streptomyces hygroscopicus, in which SpCas9 does not work well. The CRISPR-FnCas12a2 system was used to delete large fragments ranging from 21.4 to 128 kb. Additionally, the CRISPR-FnCas12a3 system employing the engineered FnCas12a mutant EP16, which recognizes a broad spectrum of PAM sequences, was used to precisely perform site mutations and insertions. The CRISPR-FnCas12a3 system addressed the limitation of TTN PAM recognition in Streptomyces strains with high GC contents. In summary, all the CRISPR-FnCas12a systems developed in this study are powerful tools for precise and multiplex genome editing in Streptomyces strains.
Highlights
Streptomyces, the largest genus of actinobacteria, has been well studied, as it contains the most prolific producers of a vast array of bioactive natural products, including antibiotics, antifungals, and anticancer agents (Baltz, 2008; Zhu et al, 2011; Cho et al, 2017; Frattaruolo et al, 2017)
To develop efficient and versatile genome editing tools in multiple Streptomyces strains, two FnCas12a systems were constructed: one based on the pWHU2653 system (Zeng et al, 2015) (CRISPR-FnCas12a1) and the other based on the pCRISPomyces-2 system (Cobb et al, 2015) (CRISPRFnCas12a2)
The FnCas12a1 system carries the selection markers codA(sm) (Dubeau et al, 2009) and aac(3)IV, the rep(pIJ101) replicon, and the B1006 terminator that terminates the transcription of the crRNA cassette, while the FnCas12a2 system carries only the selection marker aac(3)IV, the pSG5 origin of replication, and the fd terminator that terminates the transcription of the crRNA cassette (Supplementary Table 2)
Summary
Streptomyces, the largest genus of actinobacteria, has been well studied, as it contains the most prolific producers of a vast array of bioactive natural products, including antibiotics, antifungals, and anticancer agents (Baltz, 2008; Zhu et al, 2011; Cho et al, 2017; Frattaruolo et al, 2017). Efficient Genome Editing in Streptomyces increasing numbers of genetic engineering tools have been developed to explore these products (Luo et al, 2015b, 2016; Zhao et al, 2019). With the rapid development of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system (Jiang et al, 2013; Mali et al, 2013; Yan et al, 2018), effective genome editing has become increasingly easy and convenient, paving the way for us to assemble or activate uncharacterized gene clusters. NRRL S-244 (Yeo et al, 2019) and Streptomyces hygroscopicus SIPI-KF (Li et al, 2018), because of its toxicity to the hosts
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