Mechanistic and genetic basis of single-strand templated repair at Cas12a-induced DNA breaks in Chlamydomonas reinhardtii

Single-stranded oligodeoxynucleotides (ssODNs) are widely used as DNA repair templates inCRISPR/Cas precision genome editing. However, the underlying mechanisms of single-strandtemplated DNA repair (SSTR) are inadequately understood, constraining rational improve-ments to precision editing. Here we study SSTR at CRISPR/Cas12a-induced DNA double-strand breaks (DSBs) in the eukaryotic model green microalgaChlamydomonas reinhardtii.Wedemonstrate that ssODNs physically incorporate into the genome during SSTR at Cas12a-induced DSBs. This process is genetically independent of the Rad51-dependent homologousrecombination and Fanconi anemia pathways, is strongly antagonized by non-homologousend-joining, and is mediated almost entirely by the alternative end-joining enzyme poly-meraseθ. Thesefindings suggest differences in SSTR betweenC. reinhardtiiand animals. Ourwork illustrates the promising potentially ofC. reinhardtiias a model organism for studyingnuclear DNA repair.