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Crotonylation is a recently identified lysine acylation modification that was first recognized as a histone modification in 2011. This modification is characterized by the addition of a crotonyl group to lysine residues. Enriched in transcriptionally active chromatin, histone crotonylation regulates chromatin organization and gene expression. Growing evidence indicates that crotonylation participates in diverse biological processes, including cell differentiation, reproductive development, and cellular regulation. The identification of crotonylation provides new insights into the epigenetic mechanisms underlying gene expression and biological functions.