Pribylova, A.; Ponce-Lilly, C.; Molnar, A.; Fischer, L.

How cells choose among competing DNA double-strand break repair pathways, and whether the choice differs across the tree of life, remains poorly understood, limiting prediction of CRISPR editing outcomes, particularly in plants. Through a meta-analysis of 2,098 SpCas9 target sites across plants, animals, and an alga, we show that deletion patterns reflect cell-division status rather than taxonomic origin: dividing cells favour polymerase Theta-Mediated End Joining (TMEJ), whereas non-dividing cells employ a largely overlooked pathway, 5' Complementarity-Mediated End Joining (5'CMEJ). Unlike the known resection-dependent pathways, which use 3' overhangs, 5'CMEJ exploits the 5' overhangs generated by staggered SpCas9 cleavage.