The Spermidine Synthase Gene : A Novel Auxotrophic Marker for Designed by Enhanced CRISPR/Cas9 Gene Editing.

Cells

Center for Biotechnology (CeBiTec), Faculty of Biology, Bielefeld University, Universitätsstrasse 27, 33615 Bielefeld, Germany.

Published: February 2022

Biotechnological application of the green microalga hinges on the availability of selectable markers for effective expression of multiple transgenes. However, biological safety concerns limit the establishment of new antibiotic resistance genes and until today, only a few auxotrophic markers exist for . The recent improvements in gene editing via CRISPR/Cas allow directed exploration of new endogenous selectable markers. Since editing frequencies remain comparably low, a Cas9-sgRNA ribonucleoprotein (RNP) delivery protocol was strategically optimized by applying nitrogen starvation to the pre-culture, which improved successful gene edits from 10% to 66% after pre-selection. Probing the essential polyamine biosynthesis pathway, the spermidine synthase gene () is shown to be a potent selectable marker with versatile biotechnological applicability. Very low levels of spermidine (0.75 mg/L) were required to maintain normal mixotrophic and phototrophic growth in newly designed spermidine auxotrophic strains. Complementation of these strains with a synthetic gene was achieved when the mature protein was expressed in the cytosol or targeted to the chloroplast. This work highlights the potential of new selectable markers for biotechnology as well as basic research and proposes an effective pipeline for the identification of new auxotrophies in .

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8909627PMC
http://dx.doi.org/10.3390/cells11050837DOI Listing

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