Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces.

Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces.

Dissection of 3D chromosome organization in A3(2) leads to biosynthetic gene cluster overexpression.

The soil-dwelling filamentous bacteria, is widely known for its ability to produce numerous bioactive natural products. Despite many efforts toward their overproduction and reconstitution, our limited understanding of the relationship between the host's chromosome three dimension (3D) structure and the yield of the natural products escaped notice. Here, we report the 3D chromosome organization and its dynamics of the model strain, , during the different growth phases.

Improving the Precision of Base Editing by Bubble Hairpin Single Guide RNA.

Base editing is a powerful genome editing approach that enables single-nucleotide changes without double-stranded DNA breaks (DSBs). However, off-target effects as well as other undesired editings at on-target sites remain obstacles for its application. Here, we report that bubble hairpin single guide RNAs (BH-sgRNAs), which contain a hairpin structure with a bubble region on the 5' end of the guide sequence, can be efficiently applied to both cytosine base editor (CBE) and adenine base editor (ABE) and significantly decrease off-target editing without sacrificing on-target editing efficiency.

Exploration of Hygromycin B Biosynthesis Utilizing CRISPR-Cas9-Associated Base Editing.

Hygromycin B is an aminoglycoside antibiotic widely used in industry and biological research. However, most of its biosynthetic pathway has not been completely identified due to the immense difficulty in genetic manipulation of the producing strain. To address this problem, we developed an efficient system that combines clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9-associated base editing and site-specific recombination instead of conventional double-crossover-based homologous recombination.

An Fe - and α-Ketoglutarate-Dependent Halogenase Acts on Nucleotide Substrates.

While halogenated nucleosides are used as common anticancer and antiviral drugs, naturally occurring halogenated nucleosides are rare. Adechlorin (ade) is a 2'-chloro nucleoside natural product first identified from Actinomadura sp. ATCC 39365.