Base editing in <i>Streptomyces</i> with Cas9-deaminase fusions

Zhong, Zibiao; Guo, J.; Deng, Lijun; Chen, L; Wang, J; S, Li; Xu, Wentao; Deng, Zixin; Sun, Yuhui

doi:10.1101/630137

Cited by 13 publications

(16 citation statements)

References 40 publications

(43 reference statements)

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“…with a base editor architecture known as Target-AID (25)(26)(27). Whereas other CBE architectures have been used in prokaryotes as well, base editing has not been applied in the Rhizobiaceae yet (23,(28)(29)(30)(31)(32)(33).…”

Section: Significancementioning

confidence: 99%

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Rodrigues

Karimi

Impens

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Agrobacteriumspp. are important plant pathogens that are the causative agents of crown gall or hairy root disease. Their unique infection strategy depends on the delivery of part of their DNA to plant cells. Thanks to this capacity, these phytopathogens became a powerful and indispensable tool for plant genetic engineering and agricultural biotechnology. AlthoughAgrobacteriumspp. are standard tools for plant molecular biologists, current laboratory strains have remained unchanged for decades and functional gene analysis ofAgrobacteriumhas been hampered by time-consuming mutation strategies. Here, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-mediated base editing to enable the efficient introduction of targeted point mutations into the genomes of bothAgrobacterium tumefaciensandAgrobacterium rhizogenes. As an example, we generated EHA105 strains with loss-of-function mutations inrecA, which were fully functional for maize (Zea mays) transformation and confirmed the importance of RolB and RolC for hairy root development byA. rhizogenesK599. Our method is highly effective in 9 of 10 colonies after transformation, with edits in at least 80% of the cells. The genomes of EHA105 and K599 were resequenced, and genome-wide off-target analysis was applied to investigate the edited strains after curing of the base editor plasmid. The off-targets present were characteristic of Cas9-independent off-targeting and point to TC motifs as activity hotspots of the cytidine deaminase used. We anticipate that CRISPR-mediated base editing is the start of “engineering the engineer,” leading to improvedAgrobacteriumstrains for more efficient plant transformation and gene editing.

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Section: Significancementioning

confidence: 99%

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Rodrigues

Karimi

Impens

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Taking advantage of such tool, we succeeded in targeting up to 54 targets in the Mmm chromosome with a single sgRNA (Figure 4). Multiple targeting of repeated sequences has been previously reported in eukaryotic and prokaryotic systems (30, 31, 49), but this is the first time that so many target sites have been modified in bacteria. This application of CBE opens new possibilities for targeting multigene families with a limited number of sgRNA molecules.…”

Section: Discussionmentioning

confidence: 96%

Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems

Ipoutcha

Rideau

Gourgues

et al. 2022

Preprint

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Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically important livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is still a bottleneck for many mycoplasma species. Here, we demonstrate the efficacy of a CRISPR-derived genetic tool to introduce targeted mutations in three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp. mycoides. As a proof of concept, we successfully used an inducible dCas9-cytidine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was sufficiently powerful to disrupt 54 of 55 insertion sequence transposases in a single step. Whole genome sequencing showed that off-target mutations were limited and suggest that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and, most likely, the entire class Mollicutes.SignificanceMycoplasmas are minimal wall-less pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies for many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. We developed a CRISPR-derived base editor for three major pathogenic species, Mycoplasma gallisepticum, Mycoplasma bovis, and Mycoplasma mycoides subsp. mycoides. Several virulence factors were successfully targeted and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies in these economically important bacteria.

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“…Multiplex editing is expected to further accelerate strain construction [3]. Multiplex inactivation of key biosynthetic genes within a target BGC in streptomycetes can also be achieved with base editing using Cas9-deaminase fusions [30]. Last but not least, a major consideration of any genome editing strategy is that it requires the introduction of recombinant DNA, which may be challenging depending on the target actinomycete strain.…”

Section: Discussionmentioning

confidence: 99%

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

Yeo

Heng

Tan

et al. 2019

Preprint

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3Using an established CRISPR-Cas mediated genome editing technique for streptomycetes, we 4 explored the combinatorial biosynthesis potential of the auroramycin biosynthetic gene cluster in 5 Streptomyces roseoporous. Auroramycin is a potent anti-MRSA polyene macrolactam. In addition, it 6 also displays antifungal activities, which is unique among structurally similar polyene macrolactams, 7 such as incednine and silvalactam. In this work, we employed different engineering strategies to target 8 glycosylation and acylation biosynthetic machineries within its recently elucidated biosynthetic pathway. 9Six auroramycin analogs with variations in C-, N-methylation, hydroxylation and extender units 10 incorporation were produced and characterized. By comparing the bioactivity profiles of these analogs, 11we determined that unique disaccharide motif of auroramycin is essential for its antimicrobial bioactivity. 12We further demonstrated that C-methylation of the 3, 5-epi-lemonose unit, which is unique among 13 structurally similar polyene macrolactams, is key to its antifungal activity. 14 15 16 17 19 proportion of current drugs being natural product or natural product-derived [1]. Advances in genome 20 editing and synthetic biology tools, together with natural product biosynthesis knowledge accumulated 21 over the decades, allows us to better predict, design and build pathways towards the synthesis of 22 natural products [2]. Earlier, we established a rapid and efficient CRISPR-Cas9 strategy for biosynthetic 23 gene cluster (BGC) editing and activation in streptomycetes [3, 4], which opens up opportunities for 24 combinatorial biosynthesis in native streptomycete hosts [5]. Compared to chemical syntheses, 25 combinatorial engineering of native biosynthetic pathways allows us to design and biosynthesize 26 structurally complex chemical analogs without traversing difficult multi-step and possibly low-yielding 27 chemical reactions, thus facilitating elucidation of structure-activity relationships towards an optimized 28 drug lead.In this study, we describe our efforts to engineer the BGC of antimicrobial compound auroramycin [6, 1 7] and characterize its structure activity relationship (SAR). Auroramycin (1) is a polyene macrolactam 2 that is doubly glycosylated. Sugars are attached in the order of xylosamine and 3, 5-epi-lemonose to 3 the polyketide core. Compared to structurally similar natural products (Figure 1), such as the doubly 4 glycosylated incednine [8] and monoglycosylated silvalactam [9], auroramycin is the only polyene 5 macrolactam with reported antifungal activity to date. One of the main structural differences between 6 auroramycin, incednine and silvalactam is their glycosylation pattern (Figure 1). Glycosylation can 7 significantly increase the diversity and complexity of natural products and has often been shown to 8 directly and significantly impact the their bioactivity and pharmacological properties [10, 11]. As such, 9 glycodiversification is an attractive strategy to diversify and optimize bioac...

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Base editing in Streptomyces with Cas9-deaminase fusions

Cited by 13 publications

References 40 publications

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Efficient CRISPR-mediated base editing in Agrobacterium spp.

Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems

Biosynthetic engineering of the antifungal, anti-MRSA auroramycin

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