The study applied a CRISPR/Cas9 multiplex guide RNA strategy to delete entire open reading frames of four reproductive genes in Arabidopsis thaliana, achieving homozygous deletions already in the T1 generation with rates of 8.3–30%. Deletion efficiencies correlated with DeepSpCas9 prediction scores, and phenotypic analyses revealed unexpected effects of residual gene fragments on fertilization and seed development.

The study demonstrates the implementation of CRISPR/Cas9-mediated targeted mutagenesis in the orphan crop grain amaranth (Amaranthus hypochondriacus) by editing genes of the betalain biosynthesis pathway using the CasCADE modular cloning system. It addresses the bottleneck of lacking efficient stable transformation and regeneration protocols for non‑model crops, providing a reproducible workflow for climate‑resilient breeding.