Genome Engineering in Rice Using Cas9 Variants that Recognize NG PAM Sequences.

Area of Science:

• Plant biotechnology
• Genome engineering
• Molecular biology

Background:

• CRISPR/Cas9 genome editing requires specific protospacer adjacent motif (PAM) sequences, limiting its application.
• Engineered SpCas9 variants like xCas9 and SpCas9-NG offer relaxed PAM recognition, potentially overcoming these limitations.

Purpose of the Study:

• To evaluate the gene and base editing efficiencies of xCas9 and SpCas9-NG in rice.
• To assess the PAM compatibility and specificity of these variants in a crop species.

Main Methods:

• Utilized stable transgenic rice lines for CRISPR/Cas9 gene and base editing experiments.
• Tested xCas9 and SpCas9-NG with various target sites and PAM sequences.
• Analyzed editing outcomes for mutation induction and base modification.

Main Results:

• xCas9 efficiently induced mutations at NG and GAT PAM sites but showed limited base editing activity.
• SpCas9-NG demonstrated robust gene editing across diverse NG PAMs with no nucleotide preference.
• Both variants exhibited higher specificity than SpCas9 at CGG PAM sites.
• Cytosine and adenine base editors containing SpCas9-NG showed efficient editing with broadened PAM compatibility.

Conclusions:

• xCas9 and SpCas9-NG are effective tools for expanding genome editing targets in rice.
• SpCas9-NG, particularly in base editors, significantly broadens PAM compatibility for rice genome engineering.
• These engineered nucleases offer versatile solutions for crop improvement and genetic studies.