Chlamydomonas reinhardtii is a model organism for unicellular green microalgae and is widely used in basic and applied research. Nonetheless, proceeding towards synthetic biology requires a full set of manipulation techniques for inserting, removing, or editing genes. Despite recent advancements in CRISPR/Cas9, still significant limitations in producing gene knock-outs are standing, including unsatisfactory genome editing (GE) efficiency and uncontrolled DNA random insertion of antibiotic resistance markers. 

Thus, we developed an efficient DNA-free gene disruption strategy towards single and multiple genes, relying on phenotypical identification of mutants, to precisely determine its efficiency compared to marker-relying approaches and establish a new DNA-free editing tool. These results expand the toolset of GE techniques and, additionally, lead the way to future strategies to generate complex genotypes or to functionally investigate gene families.