Features affecting Cas9-induced editing efficiency and patterns in tomato: evidence from a large CRISPR dataset

Area of Science:

• Plant molecular biology
• Genome editing technologies
• Biotechnology

Background:

• CRISPR/Cas9 is vital for plant genome editing.
• Understanding sgRNA efficiency and DNA repair in plants is limited.
• Predicting sgRNA performance across species is challenging.

Purpose of the Study:

• To investigate factors influencing CRISPR/Cas9 editing efficiency in tomato.
• To analyze DNA repair outcomes and their relationship with genomic features.
• To evaluate the cross-species predictability of sgRNA design tools.

Main Methods:

• Generated a dataset of 420 sgRNAs targeting 137 tomato genes in protoplasts.
• Quantified editing efficiency, repair footprints, chromatin accessibility, and transcriptional state.
• Compared editing outcomes with genomic context and analyzed repair mechanisms.

Main Results:

• Editing efficiency correlated positively with chromatin accessibility and modestly with promoter/intron targets.
• sgRNA performance was more consistent within genes than across different genes.
• High-efficiency sgRNAs produced repair footprints suggesting microhomology-mediated end joining (MMEJ).
• Human-trained prediction models showed poor accuracy for plant sgRNA performance.

Conclusions:

• Chromatin accessibility and local genomic context significantly influence plant CRISPR/Cas9 activity.
• MMEJ appears to be a conserved repair pathway for high-efficiency editing in plants and humans.
• Developing plant-specific sgRNA design tools and understanding plant DNA repair mechanisms are crucial.