Casδ, an evolutionary transitional CRISPR system enables efficient genome editing across animals and plants

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems are vital for genome editing and DNA detection.
• Type V CRISPR-Cas systems utilize single effectors like Cas12.
• Mining microbial genomic data is crucial for discovering novel genetic tools.

Purpose of the Study:

• To identify and characterize new CRISPR-Cas effector systems.
• To evaluate the genome editing capabilities of newly discovered systems.
• To understand the evolutionary relationship of novel CRISPR-Cas nucleases.

Main Methods:

• Large-scale genomic and metagenomic data mining.
• Biochemical assays for nuclease activity.
• Genome editing experiments in human and plant cells (Oryza sativa, Zea mays).
• Structural and evolutionary analyses.

Main Results:

• Discovery of the Casδ effector superfamily (three members).
• Casδ-1 identified as an RNA-guided endonuclease with specific PAM recognition (5'-RYR-3').
• Demonstrated robust DNA cleavage and trans-cleavage activities.
• Achieved up to 60% indel rates in human cells and homozygous knockouts in rice and maize.
• Casδ identified as an evolutionary bridge between Cas12n and Type V systems.

Conclusions:

• Casδ represents a novel, compact, tracrRNA-free CRISPR system.
• Casδ enables versatile cross-kingdom genome editing.
• The C-terminal loop of Casδ is essential for its nuclease activity.
• Casδ expands the toolkit for genome engineering applications.