Efficient CRISPR Editing in Chlamydomonas

Electroporation is an established method for transforming the green alga Chlamydomonas reinhardtii. Electroporators apply short (<~100 ms) electrical pulses that create transient pores in a cell's plasma membrane. Charged molecules (DNA, RNA, proteins) in close proximity to the plasma membrane can become embedded in the membrane after these transient pores have formed [2] (link). After several hours, the charged molecules may completely cross the plasma membrane and enter the cell. Genome edit rates using electroporation for CRISPR/Cas9 have been low [3 (link),4] (link). But recently, a high-efficiency protocol has been developed to electroporate Cas9 and sgRNA expression vectors into a wall-less strain (CC-3403) of C. reinhardtii. Under optimal conditions, 16% of transformants selected for antibiotic resistance contained Cas9-generated mutations as targeted by guide RNAs [1] (link). However, the apparatus used to perform the electroporation in this efficient protocol (NEPA21 Super Electroporator, NEPA Gene Co.) is quite expensive and not widely available. The goal of this work was to determine whether more commonly available electroporators could produce the high transformation efficiencies achieved by the NEPA21.