Genome editing in plants by engineered CRISPR-Cas9 recognizing NG PAM

Area of Science:

• Molecular Biology
• Plant Science
• Biotechnology

Background:

• Streptococcus pyogenes Cas9 (SpCas9) is a key tool for genome editing.
• SpCas9 requires a specific protospacer adjacent motif (NGG) for DNA targeting.
• Expanding SpCas9's PAM compatibility is crucial for broader genome editing applications.

Purpose of the Study:

• To engineer SpCas9 variants with altered PAM specificity.
• To assess the efficacy of engineered SpCas9 variants in plant genomes.
• To develop SpCas9-based base editing systems with novel PAM recognition.

Main Methods:

• Protein engineering of SpCas9 to create SpCas9-NGv1.
• In vivo mutagenization assays in rice and Arabidopsis using SpCas9-NGv1.
• Fusion of SpCas9-NGv1 nickase with cytidine deaminase for base editing.

Main Results:

• SpCas9-NGv1 efficiently mutagenized endogenous target sites with NG PAMs in rice and Arabidopsis.
• The SpCas9-NGv1 nickase-deaminase fusion enabled precise C-to-T substitutions.
• Mutagenesis and base editing occurred near the 5' end of the target sequence.

Conclusions:

• Engineered SpCas9-NGv1 expands the targeting range of CRISPR-Cas9 in plants.
• SpCas9-NGv1 is a versatile tool for both gene knockout and base editing.
• This work provides new avenues for precise genome engineering in crops and model plants.