Genome Engineering in Plant Using an Efficient CRISPR-xCas9 Toolset With an Expanded PAM Compatibility

Area of Science:

• Plant Biotechnology
• Genome Editing
• Molecular Biology

Background:

• The CRISPR-Cas9 system is a powerful genome editing tool, but its utility is limited by the protospacer adjacent motif (PAM) requirement of the canonical Streptococcus pyogenes Cas9 (SpCas9).
• Engineered SpCas9 variants like xCas9 broaden PAM compatibility, but their application in rice, particularly for GAA and GAT PAM sites, remains underexplored.

Purpose of the Study:

• To develop an efficient CRISPR-xCas9 genome editing system for rice with expanded PAM compatibility.
• To create a novel xCas9-based cytosine base editor (CBE) for rice genome editing.
• To enhance gene mutation capabilities at various PAM sites in rice.

Main Methods:

• Utilized tRNA and enhanced sgRNA (esgRNA) to construct an efficient CRISPR-xCas9 system.
• Applied the CRISPR-xCas9 system to target NG, GAA, GAT, and GAG PAM sites in rice.
• Developed and tested an xCas9-based cytosine base editor targeting NG and GA PAM sites.

Main Results:

• Successfully generated a CRISPR-xCas9 genome editing system capable of mutating genes at NG, GAA, GAT, and GAG PAM sites in rice.
• Developed a functional xCas9-based cytosine base editor for editing NG and GA PAM sites in rice.
• Demonstrated the system's efficiency in generating edited rice plants, overcoming previous limitations.

Conclusions:

• The developed CRISPR-xCas9 system and xCas9-based CBE significantly expand the targeting range for genome editing in rice.
• These novel tools provide powerful resources for future rice research, breeding, and potentially other plant species.
• The ability to edit diverse PAM sites, including GAG, GAA, and GAT, enhances the versatility of CRISPR technology in plants.