Improving iSpyMacCas9 multiplex genome editing in rice by CRISPR-combo-mediated BBM1 activation

Area of Science:

• Plant biotechnology
• Genome editing technologies

Background:

• CRISPR-Combo enables simultaneous gene editing and activation in plants.
• SpCas9 in CRISPR-Combo is limited by AT-rich genomic loci, hindering promoter targeting.

Purpose of the Study:

• To expand CRISPR-Combo by incorporating Cas12b and iSpyMacCas9 for enhanced genome editing and transcriptional activation.
• To assess the efficiency of these expanded systems in hormone-free rice regeneration via OsBBM1 activation and gene knockout.

Main Methods:

• Developed and tested Cas12b-Combo and iSpyMacCas9-Combo systems in rice.
• Evaluated OsBBM1 transcriptional activation and genome editing efficiency.
• Assessed hormone-free plant regeneration rates.

Main Results:

• Cas12b-Combo showed mild OsBBM1 upregulation (~3-fold) without impacting editing efficiency.
• iSpyMacCas9-Combo achieved significant OsBBM1 activation (~12-fold), leading to 42% hormone-free regeneration.
• iSpyMacCas9-Combo demonstrated superior genome editing efficiency, including multiplex editing, compared to standard iSpyMacCas9.

Conclusions:

• iSpyMacCas9-Combo is a highly efficient system for rice genome editing, particularly at challenging AT-rich sites.
• The expanded CRISPR-Combo toolkit, especially iSpyMacCas9-Combo, broadens targeting scope and enhances genome editing efficacy through endogenous gene activation.