Novel genome editing technique in Chlamydomonas reinhardtii and its underlying DNA repair pathway
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Date
04/10/2022Item status
Restricted AccessEmbargo end date
04/10/2023Author
Ferenczi, Aron
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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.