Versatile genome editing using Type I-E CRISPR-Cas3 in rice.

Area of Science:

• Plant biotechnology
• Molecular biology
• Genome editing

Background:

• Type I-E CRISPR-Cas3 from Escherichia coli (Eco CRISPR-Cas3) facilitates large deletions in mammalian genome editing.
• Implementing Eco CRISPR-Cas3 in plants is complex due to the simultaneous expression requirement of seven components.
• Previous plant applications were limited to maize protoplasts, with no mutant plants generated.

Purpose of the Study:

• To develop and validate an Eco CRISPR-Cas3 genome editing system for rice.
• To assess the efficiency of generating deletions and base editing in rice.
• To investigate the inheritance of induced mutations in rice.

Main Methods:

• Agrobacterium-mediated transformation was used to introduce the Eco CRISPR-Cas3 system into rice.
• Polymerase Chain Reaction (PCR) and droplet digital PCR (ddPCR) were employed to detect and quantify gene deletions.
• Regenerated rice plants and their progenies were analyzed via sequencing to confirm mutations and inheritance.

Main Results:

• Deletions were detected in 39-71% of transformed rice calli.
• Allele frequencies for large deletions (up to 7.0 kb upstream of PAM) ranged from 21-61%.
• Sequencing confirmed deletions of 0.1-7.2 kb, including novel alleles with insertions and inversions, and demonstrated C to T base editing.

Conclusions:

• The Eco CRISPR-Cas3 system is effective for generating large deletions and base editing in rice.
• This system facilitates gene knockout, deletion, base editing, and genome rearrangement in plants.
• Eco CRISPR-Cas3 represents a promising tool for advancing plant genome editing applications.