Modification of a Chlamydomonas reinhardtii CRISPR/Cas9 transformation protocol for use with widely available electroporation equipment

Abstract

A recently reported protocol demonstrates efficient CRISPR/Cas9 gene editing of Chlamydomonas reinhardtii [1]. The published protocol demonstrates transformation and editing of a wall-less strain of C. reinhardtii using plasmid encoded Cas9 and sgRNA. However, the published protocol utilizes a complex electroporation waveform that cannot be generated by most electroporation systems. It is unknown whether transformation via this complex electroporation waveform is essential for high efficiency of Cas9 edits, perhaps by optimizing Cas9 or guide RNA gene expression or incorporation into the genome. We demonstrate that a simple electroporation waveform can deliver plasmid encoded CRISPR/Cas9 into and edit the genome of a wall-less strain of C. reinhardtii as efficiently as the more complex waveform. Our modified electroporation protocol makes the plasmid based CRISPR/Cas9 genome editing method accessible to a greater number of Chlamydomonas researchers. • Our protocol uses a simple electroporation waveform to replace a complex waveform used to achieve efficient CRISPR/Cas9 gene editing in a wall-less strain of Chlamydomonas reinhardtii. • We also increased concentration of plasmids to maintain high gene editing efficiency. • We minimized modifications to other steps of the original protocol.

Keywords

• Electroporation
• Exponential pulse
• CRISPR/Cas9 genome editing
• Plasmid encoded Cas9