Integrating transcriptomics and high-throughput gene editing uncovers shoot apical meristem regulators in <i>Brassica napus</i>

The shoot apical meristem (SAM) determines plant architecture, but the key components of its regulatory network remain elusive in rapeseed (Brassica napus L.). Here, we integrated transcriptomic profiling of 3 multilocular silique mutants (Bnaclv1, Bnaclv2, and Bnaclv3) across key SAM development stages (IM, stage6, and stage8) with large-scale CRISPR/Cas9 functional screening to identify regulators of SAM maintenance. Differential gene expression and GO enrichment highlighted genes significantly associated with meristem development processes. Weighted gene co-expression network analysis of stage-specific transcriptomes identified 42 candidate genes potentially related to SAM development. To enable systematic functional screening, we established a high-throughput multiplex CRISPR/Cas9 pipeline, simultaneously targeting 198 sites across 42 candidate genes through optimized sgRNA design and pooled transformation. We successfully obtained mutants for 25 genes with homozygous mutants for 9 genes. Phenotypic analysis demonstrated that mutants of BnaSCPL family genes (SCPL29, SCPL44, and SCPL45) exhibited a multi-stem phenotype and disrupted SAM organization. Mechanistic studies revealed that BnaSCPL mutations disrupt the canonical CLV3/WUS feedback loop, uncovering their roles in SAM homeostasis. Additionally, knockout of BnaLFY homologs caused permanent vegetative state and sterility, demonstrating their conserved role in floral meristem identity in Brassica napus. Collectively, our study not only elucidates the critical function of BnaSCPLs in SAM maintenance but also establishes a regulatory framework for understanding meristem phase transitions in B. napus, providing potential targets for crop architecture improvement.