The full Monty: CRISPR protocols for efficient engineering in Streptomyces

Monday 13 Jul 20

Contact

Yaojun Tong
Researcher
DTU Biosustain
+45 24 89 35 04

Contact

Tilmann Weber
Professor
DTU Biosustain
+45 24 89 61 32

A comprehensive collection of CRISPR tools and protocols for engineering Streptomyces is now available in Nature Protocols.

In the quest for new antibiotics with new modes of action, researchers often look towards Streptomyces bacteria, which have previously proved their worth in terms of harboring potent and valuable anti-microbial compounds. Currently, more than 70 % of our antibiotics used in clinic are derived from these organisms.

 

In order to find new antibiotics and produce them in quantities large enough to study, engineering of Streptomyces is important. And here comes the trouble – standard genome editing tools are a major bottleneck to achieve this.

 

Therefore, Professors Sang Yup Lee and Tilmann Weber and their team of researchers from The Novo Nordisk Foundation Center for Biosustainability at DTU have worked hard to produce a toolbox for genome editing in Streptomyces using the relatively new CRISPR-approaches. The tools have now been written into standard protocols and published in Nature Protocols together with a blog "behind the paper" in Springer Nature Protocols and Methods Community.

 

"We are very happy and proud that we can now share our tools along with a detailed protocol with others, and we hope that it will become a lot easier for others to use our methods in their own labs"
Tilmann Weber, Professor at DTU Biosustain

“We are very happy and proud that we can now share our tools along with a detailed protocol with others, and we hope that it will become a lot easier for others to use our methods in their own labs,” says Tilmann Weber.

 

The good, the bad, the CRISPR-BEST

One of the tools are called CRISPR-BEST and gives the opportunity, amongst other things, to inactivate a gene of interest without risking serious chromosomal deletions or rearrangement, which has previously been a major problem.

 

“For systematic metabolic engineering of Actinobacteria, including Streptomyces, only few genetic tools exist that have the required throughput and scalability – so the fact that we now have a new toolkit is an advantage in the discovery process,” says Researcher at the Novo Nordisk Foundation Center for Biosustainability at DTU Yaojun Tong, who is main-engineer behind the entire CRISPR-toolkit.

 

Alarming problems require focused efforts

According to WHO, currently, at least 700,000 people die each year due to drug-resistant diseases, including 230,000 people who die from multidrug-resistant tuberculosis. WHO also underlines that ‘more and more common diseases, including respiratory tract infections, sexually transmitted infections and urinary tract infections, are untreatable.’ Therefore, finding new antibiotics with novel modes of action is pressing.

 

According to Professor Tilmann Weber, developing new antimicrobial drugs is an international task, which requires as many talented people working to solve the problem as possible:

 

“Like we see in the current COVID-19 pandemic, a lot of focus on a disease can really spark a cascade of solutions. The problem of antimicrobial resistance may not be as visible as COVID-19 and may not seem equally urgent – but it is a real threat that I personally hope we find a solution to before the problem of antibiotic resistance becomes too visible and urgent,” he concludes.

About the CRISPR toolbox for Streptomyces

  • The "classical" CRISPR-Cas9 system developed here (pCRISPR-Cas9, Addgene: 125686) can be used to generate deletion libraries targeting at an editing site defined by the single 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 activities of target genes
  • To further address the concerns of double-stranded breaks (DSB), the CRISPR-BEST (Base Editing SysTem) has been built 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.
  • Along with the set of CRISPR tools, the researchers have the sgRNA design software CRISPy-web (https://crispy.secondarymetabolites.org), which supports all the tools mentioned above. It can facilitate the use of the new CRISPR toolkit described here.

 

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