Developing a new efficient CRISPR-Cas9 base Homology Directed Repair oriented gene editing plasmid for Streptomyces

The CRISPR-Cas9 editing mechanism revolutionized genetic engineering, providing a precise and versatile tool for editing the genomes of virtually any organism. Streptomyces are soil dwelling bacteria with a complex life cycle and a reputation for producing a wide array of bioactive compounds, including many clinically important antibiotics. Despite their significance, genetic manipulation in Streptomyces has traditionally been challenging. Their complex and large genomes, slow growth, and the presence of multiple, sometimes redundant, biosynthetic gene clusters made traditional methods of mutagenesis laborious and time-consuming. The implementation of CRISPR-Cas9 in Streptomyces therefore has a profound impact on both basic research and industrial applications. In particular, it is important for the development of new antibiotics at a time when rising antibiotic resistance is growing into a global health crisis. This study sought to develop a new efficient CRISPR-Cas9 gene editing plasmid that works based on the homology directed repair system in Streptomyces. The fundamental goal of the study is to characterise the phenotype of a deletion mutant in the gene cluster, SCO6357-53, by using the newly developed CRISPR-Cas9 plasmid. Previous study has shown that SCO6357-53 is a suspected teicoplanin tolerance/resistance cluster in Streptomyces coelicolor. A natural product glycopeptide, teicoplanin, is an important alternative front-line therapy to vancomycin for the treatment of Gram-positive nosocomial infections but, unlike vancomycin, its mechanism of resistance is not clearly understood. While this study sought to characterise this cluster, an inability to create the new CRISPR-Cas9 based editing plasmid prevented any genetic manipulations to be carried out. Extensive plasmid DNA analysis using restriction enzyme digestion, PCR and sequencing was conducted. An initial inability to produce the desired plasmid led to a reconfiguring of the DNA assembly, however this second method also failed to produce the desired resulting plasmid. Whole plasmid DNA sequencing analysis revealed that a range of different unexpected rearrangements of DNA occurred, with either the loss of DNA segments which are functionally vital or a gain of DNA due to an unexpected insertion of external DNA fragments in various loci in the reconfigured plasmid. Although additional unwanted DNA fragments were present, one plasmid sample seemed to preserve all functionally vital regions for the HDR oriented targeted gene editing, so further cloning based on Gibson assembly was undertaken. However, unfortunately this also failed to obtain a desired product. Thus, an inability to construct the required HDR oriented gene editing plasmid, pCRISPR-Cas9KM, meant that further investigation into the teicoplanin resistance cluster could not be carried out. In light of this, further advancements into generating this actinomycete-codon optimised genome editing tool needs to be carried out. By investigating the knockout of this teicoplanin resistance-conferring cluster, further insights into the associated resistance mechanisms can be determined. Gaining insights into these resistance mechanisms will provide a fundamental keystone in tackling the rising Anti-Microbial Resistance crisis. Through understanding these mechanisms, current antibiotics can be developed in a way that circumvent the arising bacterial resistance mechanisms, thus helping to alleviate the increasingly prevalent AMR crisis.

Grantor dc:publisher

Oxford Brookes University

Author dc:creator

• Murray, Kate

Contributors dc:contributor

• Hong, Hee-Jeon

Statement dc:rights

• All rights reserved

Language dc:language

en

DOI dc:identifier

https://doi.org/10.24384/n48h-4938

OAI identifier oai:identifier

tle:12b77752-fa09-4042-a98b-e45073e7d45e:d6bd9758-527a-46cd-bfe2-c433766e8fca:1