Base editing in rice using nuclease-deactivated CRISPR/Cas-SF01

Area of Science:

• Plant Molecular Biology
• Genetics and Genomics
• Biotechnology

Background:

• CRISPR-Cas systems have revolutionized genome editing.
• Base editing offers precise single nucleotide modification without double-strand breaks.
• Efficient base editing in rice is crucial for crop improvement.

Purpose of the Study:

• To evaluate the efficacy of dCas-SF01 for adenine and cytosine base editing in rice.
• To develop and test a novel dCas-SF01 variant for expanded PAM compatibility.
• To assess the efficiency of base editing at multiple loci in rice.

Main Methods:

• Utilized the dCas-SF01 base editor and the 35S-CmYLCV-U6 composite promoter.
• Introduced targeted base substitutions at multiple genomic sites in rice.
• Employed a protospacer adjacent motif (PAM)-relaxed SF01-IKRR variant for broader PAM recognition.

Main Results:

• Achieved successful targeted base substitutions in rice with an average editing efficiency of 33.3%.
• Demonstrated the capability of dCas-SF01 for both adenine and cytosine base editing.
• The SF01-IKRR variant enabled base editing with 5'-NTN PAMs, significantly expanding targeting scope.

Conclusions:

• dCas-SF01 is an effective tool for precise base editing in rice.
• The PAM-relaxed variant broadens the applicability of base editing in rice genome engineering.
• These advancements hold promise for accelerating rice genetic improvement and functional genomics studies.