Genetius

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 reveals that a set of REPRODUCTIVE MERISTEM (REM) transcription factors, termed RIMs, are essential for directing RNA‑directed DNA methylation (RdDM) to CLSY3 targets in a sex‑specific manner in Arabidopsis reproductive tissues. Disruption of RIM DNA‑binding domains or their target motifs abolishes RdDM at these loci, demonstrating that genetic cues can guide de novo methylation patterns.

The study identifies four Arabidopsis REM transcription factors (VDD, VAL, REM12, REM13) that bind specific DNA sequences and, together with GDE1, recruit RNA polymerase IV to produce 24‑nt siRNAs that direct DNA methylation at designated loci. Loss of GDE1 causes Pol IV complexes to relocalize to sites bound by REM8, indicating that REM proteins provide sequence‑specific cues for epigenetic patterning.