Efficient Multiplex Genome Editing in Streptomyces via Engineered CRISPR-Cas12a Systems

Area of Science:

• Microbiology
• Molecular Biology
• Biotechnology

Background:

• Streptomyces strains are prolific producers of valuable natural products, with numerous biosynthetic gene clusters identified through genome sequencing.
• Existing CRISPR-Cas9 gene editing tools face limitations in certain Streptomyces strains and have restricted protospacer adjacent motif recognition, hindering precise genetic manipulation.
• The need for versatile and efficient genome editing tools is critical for harnessing the potential of Streptomyces for drug discovery.

Purpose of the Study:

• To develop novel CRISPR-FnCas12a systems for advanced genome editing in Streptomyces.
• To address the limitations of CRISPR-Cas9 in specific industrial and newly discovered Streptomyces strains.
• To enhance the precision and scope of genome editing for manipulating biosynthetic pathways in Streptomyces.

Main Methods:

• Development and application of three distinct CRISPR-FnCas12a systems (CRISPR-FnCas12a1, CRISPR-FnCas12a2, and CRISPR-FnCas12a3).
• CRISPR-FnCas12a1 was tested in Streptomyces hygroscopicus, an industrial strain where SpCas9 is less effective.
• CRISPR-FnCas12a2 was utilized for large DNA fragment deletion (21.4–128 kb).
• CRISPR-FnCas12a3, using an engineered FnCas12a mutant (EP16), was employed for precise site mutations and insertions, overcoming TTN PAM limitations in high GC content strains.

Main Results:

• The CRISPR-FnCas12a1 system demonstrated high efficiency in the industrial strain Streptomyces hygroscopicus.
• The CRISPR-FnCas12a2 system successfully deleted large genomic fragments, showcasing its utility for significant pathway engineering.
• The CRISPR-FnCas12a3 system, with its broad PAM recognition, enabled precise gene editing, including overcoming GC-rich sequence challenges.
• All developed CRISPR-FnCas12a systems proved effective for multiplex genome editing in various Streptomyces strains.

Conclusions:

• The three developed CRISPR-FnCas12a systems offer powerful and versatile solutions for precise genome editing in Streptomyces.
• These systems expand the toolkit for genetic manipulation of Streptomyces, facilitating the exploration of biosynthetic gene clusters.
• The advancements reported are crucial for accelerating the discovery and development of novel clinical drugs from Streptomyces natural products.