Programmable site-specific nucleases, such as the CRISPR/Cas9 ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas. However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous double-stranded DNA (dsDNA) encoding a selectable marker gene. Repair of the double-stranded DNA breaks induced by the ribonucleoproteins is usually accompanied by

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... ic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. In this study, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker) - in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precisely, scarless edited FTSY or WDTC1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonas acetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways or structures.