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:

• CRISPR-Cas9 is a powerful genome editing tool but is limited by its protospacer adjacent motif (PAM) requirement.
• The canonical Streptococcus pyogenes Cas9 (SpCas9) typically requires an NGG PAM, restricting targetable sites.
• Engineered variants like xCas9 offer broader PAM compatibility, but their efficiency in plants, especially for specific PAMs, remains underexplored.

Purpose of the Study:

• To develop an efficient CRISPR-xCas9 genome editing system for rice with expanded PAM compatibility.
• To generate knockout rice plants for GAA and GAT PAM sites, which have been challenging.
• To create an xCas9-based cytosine base editor (CBE) for rice genome editing.

Main Methods:

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

Main Results:

• Successfully generated an efficient CRISPR-xCas9 system for rice, enabling mutations at NG, GAA, GAT, and GAG PAM sites.
• Achieved gene mutations at previously difficult-to-target GAA and GAT PAM sites in rice.
• Developed a functional xCas9-based cytosine base editor for rice, expanding editing capabilities.

Conclusions:

• The developed CRISPR-xCas9 system significantly broadens the scope of genome editing in rice.
• These novel tools are valuable for future rice research, crop improvement, and breeding programs.
• The system's applicability may extend to other plant species, advancing agricultural biotechnology.