The BEST is coming: Streptomyces natural products meet CRISPR toolkit

A CRISPR-based, highly efficient genome editing toolkit containing seven sub-systems for Streptomyces has been developed, which would much facilitate the bioactive natural products discovery in streptomycetes.

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Written by Yaojun Tong and Tilmann Weber.

We thank Anne Wärme Lykke for proofreading of this blog post.

Global medicine shortages have become a complicated public health issue as critically illustrated by the current COVID-19 crisis. It is, however, noteworthy that besides the shortage of antiviral drugs and vaccines there also is a lack of developing incentives for novel antibiotics. Natural products produced by bacteria and fungi are the most important resources for finding lead compounds with a potential for antimicrobial drugs. For example, more than 70 % of our antibiotics currenly used in clinic are from natural products of a group of gram-positive bacteria Streptomyces. Although these organisms have been intensively studied for over 70 years, analyzing their genome sequences tells us that each Streptomyces strain usually codes for more than 25 biosynthetic pathways for natural products – a huge and yet untapped resource for novel compounds. Hopefully, and most likely, some of these will become our new weapons of the future to address the increasing problems with antibiotic resistance. But unfortunately, the organism harboring these promising products are relatively difficult to be genetically engineered, which complicates the discovery process.

To address this challenge, we developed various CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic manipulation approaches in streptomycetes (Figure 1) that makes it possible to engineer these bacteria. All in all, these tools allow for both directed insertions/deletions without risking cell death due to large chromosomal deletions or rearrangements. Here is how the tools work: the “classical” CRISPR-Cas9 system (pCRISPR-Cas9, Addgene: 125686) can be used to generate deletion libraries targeting at an editing site defined by the singe guide RNA; an optimized version (pCRISPR-Cas9-ScaligD, Addgene: 125688). In addition, this method reconstitutes the non-homologous end joining (NHEJ) DNA repair activity of Streptomyces allowing CRISPR editing comparable to eukaryotic applications. Furthermore, with pCRISPR-dCas9 (Addgene: 125687), the toolkit contains a nuclease-inactivated variant of Cas9 that can be used for CRISPRi (CRISPR interference) to modulate transcriptional activity of target genes.

One remaining challenge with all mutagenesis procedures for streptomycetes is the observation that in some cases the introduction of DNA double strand breaks (DSBs) by Cas9 or other means spontaneously can lead to large unwanted deletions or chromosome rearrangements.

To further address this concerns, we have established the CRISPR-BEST (Base Editing SysTem), by fusing a cytidine- (CRISPR-cBEST, Addgene: 125689) or an adenosine- (CRISPR-aBEST, Addgene: 131464) deaminase to a Cas9 nickase (Cas9n, it can only break one DNA strand). Targeted by an sgRNA, CRISPR-cBEST can efficiently convert C:G basepairs to T:A basespair, and CRISPR-aBEST can convert A:T basepairs to G:C basepairs at nearly single-nucleotide-resolution. As CRISPR-BEST does not require DNA DSB to engineer the target DNA, the genome wide off-target effects are much reduced. Some very usful applications are (not limited to): inactivating a gene of interest by converting a codon to a stop codon; in vivo engineering target proteins or promoter sequences by exchanging codons;

By using a Csy4 based sgRNA processing machinery, the system furthermore multiplexed genome editing of several target sequences at one time.

Along with the set of CRISPR tools, we host the sgRNA design tool CRISPy-web (

https://crispy.secondarymetabolites.org), which supports all the tools mentioned above. It can much facilitate the use of our CRISPR toolkit.

Figure 1. A summary of CRISPR toolkit for Streptomyces.

In order to facilitate researchers world-wide to use our CRISPR toolkit, we wrote a step-by-step protocol, which can be found here.

A simple workflow of using the CRISPR toolkit is illustrated in Figure 2.

Figure 2. A simple workflow of using the CRISPR toolkit for Streptomyces.

a. in silico design of sgRNA, primers, and editing templates.

b. construct the designed CRISPR plasmid.

c. validate the CRISPR plasmid.

d. transfer the CRISPR plasmid into the target streptomycetes by conjugation.

e. validate the correctly edited target streptomycetes, and make glycerol stock for long-term storage.

We hope that our CRISPR toolkit will contribute to and accelerate metabolic engineering of streptomycetes and with that support basic research on this fascinating group of bacteria and boost the discovery process of novel antimicrobials.

 

 

Yaojun Tong

Researcher, The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark

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