Novel genome editing technique in Chlamydomonas reinhardtii and its underlying DNA repair pathway

Abstract

The green microalga Chlamydomonas reinhardtii is an important model organism, with potential industrial biotechnology applications, including potential for vaccine production (e.g., TransAlgae, Israel) and vegan protein (e.g., Triton Algae Innovations, U.S.). However, gene editing techniques were lacking for this organism when this thesis started, constraining reverse genetics and industrial cell line development. In Chapters 2 and 3, we developed an efficient, transgene-free, CRISPR-based precision genome editing technique for C. reinhardtii, capable of editing cells with double-digit percentage efficiencies as a proportion of all viable cells (without positive selection). We find that single-stranded oligodeoxynucleotides (ssODN or ssDNA), used to instruct the desired edits, act as gene editing ‘efficiency boosters’ in C. reinhardtii unlike in any other organism to my knowledge. In Chapter 4, we compare CRISPR nucleases Cas12 and Cas9 in C. reinhardtii.

Finally, in Chapter 5, we examine the mechanistic and molecular mechanism of the relatively poorly understood DNA repair pathway underpinning ssODN-mediated repair, called single-strand templated repair (SSTR). We find differences in SSTR compared to metazoans, fundamentally advancing our understanding of this pathway in a plant (Viridiplantae) system. Our findings also suggest ways to optimize ssODN-mediated editing further. Taken together, our work demonstrates the potential of C. reinhardtii as a tractable model organism for developing and optimizing gene editing methods and for studying DNA repair.