The authors used a bottom‑up thermodynamic modelling framework to investigate how plants decode calcium signals, starting from Ca2+ binding to EF‑hand proteins and extending to higher‑order decoding modules. They identified six universal Ca2+-decoding modules that can explain variations in calcium sensitivity among kinases and provide a theoretical basis for interpreting calcium signal amplitude and frequency in plant cells.

The study benchmarked over 20 web‑based gRNA on‑target efficiency prediction tools against an experimental plant CRISPR editing dataset, finding several machine‑learning based tools whose scores strongly correlated with observed InDel frequencies. Additionally, the performance of popular platforms such as CRISPOR and CRISPR‑P was assessed, offering guidance for improved gRNA design in plant genome editing.

Populus VariantDB v3.2 is a searchable database that catalogs sequence polymorphisms across heterozygous poplar genomes, enabling precise design of guide RNAs and primers for CRISPR genome editing in outcrossing tree species. The tool accepts short sequence inputs, supports multiple Populus genomes (including hybrids), and provides customizable search parameters to improve gRNA/primer design accuracy. Its platform‑agnostic and expandable architecture makes it broadly applicable to sequence‑sensitive functional genomics studies.