PAM-less plant genome editing using a CRISPR-SpRY toolbox.

Area of Science:

• Plant biotechnology
• Genome engineering
• Molecular biology

Background:

• CRISPR-Cas9, -Cas12a, and -Cas12b systems have advanced plant research.
• Cas nuclease DNA targeting is limited by protospacer adjacent motifs (PAMs).
• The SpCas9 NGG PAM restricts targeting to GC-rich regions in plants.

Purpose of the Study:

• To demonstrate genome editing at relaxed PAM sites in plants using an engineered SpRY Cas9 variant.
• To develop and apply SpRY-based cytosine and adenine base editors for plant genome engineering.
• To overcome PAM restriction barriers in plant CRISPR applications.

Main Methods:

• Utilized an engineered SpRY Cas9 variant for genome editing in rice and Dahurian larch.
• Applied SpRY for targeted mutagenesis via non-homologous end joining (NHEJ) in rice.
• Developed SpRY-based cytosine and adenine base editors through directed evolution.

Main Results:

• Achieved highly efficient targeted mutagenesis at relaxed PAM sites in Dahurian larch protoplasts and rice transgenic lines.
• Developed a SpRY-based cytosine base editor for directed evolution of herbicide-resistant OsALS alleles in rice.
• Created a SpRY-adenine base editor (SpRY-ABE8e) achieving up to 79% editing efficiency in rice plants.
• Demonstrated SpRY's ability to enable DNA editing in a PAM-less fashion, breaking PAM restriction barriers.

Conclusions:

• The SpRY toolbox significantly expands the targeting scope of CRISPR-Cas9 in plant genome engineering.
• SpRY enables efficient base editing for trait development, such as herbicide resistance in rice.
• Potential for secondary off-target effects from de novo sgRNA generation necessitates sophisticated sgRNA design programs for SpRY applications.