crispy

crispy enables DSB-free base editing in streptomycetes by integrating CRISPR-BEST base editors (CRISPR-cBEST and CRISPR-aBEST) with CRISPy-web protospacer identification to perform precise single-nucleotide conversions (C:G to T:A and A:T to G:C) and targeted STOP codon introduction.

Key Features:

• DSB-Free Genome Editing: CRISPR-BEST eliminates the need for DNA double-strand breaks (DSBs), mitigating genome instability and Cas9-associated toxicity observed with traditional CRISPR-Cas9 systems.
• Single-Nucleotide Resolution: Two base editors, cytidine deaminase-based CRISPR-cBEST and adenosine deaminase-based CRISPR-aBEST, enable precise conversion of C:G to T:A and A:T to G:C within defined target windows.
• High Fidelity and Specificity: CRISPR-BEST has been validated for high-fidelity editing with minimal reported off-target effects.
• Multiplexed Editing Capability: Multiplexed genome editing is supported using a single plasmid and a Csy4-based sgRNA processing machinery to express multiple sgRNAs.
• Application in Nonmodel Actinomycetes: CRISPR-BEST has been applied to nonmodel actinomycetes such as Streptomyces collinus Tü365 to inactivate duplicated biosynthetic genes by introducing STOP codons where DSB-based methods were ineffective.
• Protospacer Identification via CRISPy-web: CRISPy-web has been updated to streamline protospacer identification for sgRNA design specifically for CRISPR-BEST applications.

Scientific Applications:

• Streptomycetes genetics: Precise, DSB-free edits enable functional interrogation of genes in streptomycetes involved in secondary metabolism and natural product biosynthesis.
• Functional Genomics: Single-nucleotide and STOP-codon edits facilitate loss-of-function and allele-specific studies for gene function characterization.
• Metabolic Engineering and Natural Product Development: Targeted base edits support pathway optimization and generation of novel derivatives in antibiotic and secondary metabolite research.

Methodology:

CRISPy-web performs protospacer identification for sgRNA design tailored to CRISPR-BEST base editors.