Efficient methods for multiple types of precise gene‐editing in Chlamydomonas

SUMMARY Precise gene‐editing using CRISPR/Cas9 technology remains a long‐standing challenge, especially for genes with low expression and no selectable phenotypes in Chlamydomonas reinhardtii, a classic model for photosynthesis and cilia research. Here, we developed a multi‐type and precise genetic manipulation method in which a DNA break was generated by Cas9 nuclease and the repair was mediated using a homologous DNA template. The efficacy of this method was demonstrated for several types of gene editing, including inactivation of two low‐expression genes (CrTET1 and CrKU80), the introduction of a FLAG‐HA epitope tag into VIPP1, IFT46, CrTET1 and CrKU80 genes, and placing a YFP tag into VIPP1 and IFT46 for live‐cell imaging. We also successfully performed a single amino acid substitution for the FLA3, FLA10 and FTSY genes, and documented the attainment of the anticipated phenotypes. Lastly, we demonstrated that precise fragment deletion from the 3′‐UTR of MAA7 and VIPP1 resulted in a stable knock‐down effect. Overall, our study has established efficient methods for multiple types of precise gene editing in Chlamydomonas, enabling substitution, insertion and deletion at the base resolution, thus improving the potential of this alga in both basic research and industrial applications. Significance Statement Efficient precision gene‐editing system was established in Chlamydomonas, including target gene inactivation, single amino acid substitution, knock‐in of an epitope or a YFP tag, and the precise deletion of a DNA fragment in the genome. The methods established in this study will facilitate generating multiple types of genetically modified strains, re‐constructing the metabolism pathway for producing the bio‐fuel and high‐value algal compounds, and modifying the structure of the cell wall for bio‐refinery.