With the establishment of the CRISPR-Cas9 molecular tool as a DNA editing system in 2012, the handling of gene editing experiments was strongly facilitated pushing reverse genetics approaches forward in many organisms. These new gene editing technologies also drastically increased the possibilities for design-driven synthetic biology. Here, we describe a protocol for gene editing in the green algae Chlamydomonas reinhardtii using preassembled CRISPR-Cas9 ribonucleoproteins.The three sections of the protocol guide through a complete gene editing experiment, starting with the experimental design and the choice of suitable CRISPR target sites and how to perform a Cas9 in vitro test digestion. The second part covers the transformation of algal cells with Cas9 RNPs using electroporation. In the last part, the PCR-based screening for mutants and isolation of clones is explained.
Keywords: CRISPR/Cas9; Chlamydomonas reinhardtii; Gene editing; Gene targeting; Green algae; Homologous recombination (HR); Homology-directed repair (HDR); Microalgae; RNP; Ribonucleoproteins; S. pyogenes; SpCas9; Synthetic biology.
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