CRISPR/dCas-mediated gene activation toolkit development and its application for parthenogenesis induction in maize

Area of Science:

• Plant science
• Molecular biology
• Biotechnology

Background:

• Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems are adaptable for gene transcription modulation.
• Apomixis is crucial for the seed industry, enabling clonal seed production with consistent genetic backgrounds.
• Parthenogenesis, or asexual reproduction, is a key target for enhancing plant breeding strategies.

Purpose of the Study:

• To develop and apply a novel CRISPR activation (CRISPRa) toolkit for targeted gene activation in maize.
• To investigate the potential of CRISPRa in inducing parthenogenesis by targeting the maize BABY BOOM2 (ZmBBM2) gene.
• To establish a precise gene-activation technology for specific cell types in vivo.

Main Methods:

• Engineered a CRISPR/dCas9-based toolkit utilizing dCas9-VP64 and MS2-p65-HSF1 effectors for high gene activation.
• Applied in vivo CRISPRa to target the ZmBBM2 gene, identified as a critical fertilization checkpoint in maize.
• Utilized transcript detection to confirm ZmBBM2 expression specifically in egg cells.

Main Results:

• The CRISPRa toolkit demonstrated high specificity and activation capability in targeting ZmBBM2.
• ZmBBM2 transcripts were exclusively detected in maize egg cells, not in other maternal gametic cells.
• In vivo activation of ZmBBM2 in egg cells resulted in maternal cell-autonomous parthenogenesis, producing haploid seeds.

Conclusions:

• Developed a highly specific CRISPRa technology for targeted gene activation within specific plant cells.
• Successfully demonstrated the induction of parthenogenesis in maize using CRISPRa to activate ZmBBM2 in egg cells.
• This research offers a valuable tool for parthenogenesis induction, with significant implications for seed industry breeding programs.