Genome editing of diatoms using CRISPR/Cas9 technology - Creation of LPA2 and LPA3 mutant Phaeodactylum tricornutum strains
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- Institutt for biologi 
The diatom (Bacillaryophycea) Phaeodactylum tricornutum is a photosynthetic, unicellular eukaryote phytoplankton, living in freshwaters and oceans. Diatoms are nutritionally suitable as feedstock for the marine culture industry, but the biomass density in a culture is limited by the availability of light. A suggested way to optimize growth and productivity in mass cultures under bright sunlight conditions is to create truncated light-harvesting antenna (TLA) mutants. Knocking out genes of the chloroplast signal recognition particle (CpSRP) pathway has resulted in strongly reduced antennae size and improved photosynthetic performance in high density cultures, however some light-harvesting antenna proteins are still inserted and assembled in the thylakoid membrane. Existing literature on the role of the low PSII accumulation 2 (LPA2) and low PSII accumulation 3 (LPA3) proteins in higher plants at the time of the project start-up, suggested that both LPA2 and LPA3 interact with a protein in the CpSRP pathway (Alb3). These genes were therefore suitable for investigation, in the work to identify a mutant with the smallest possible light harvesting antennae. The newly developed CRISPR-Cas technology facilitates high-precision genome editing. The CRISPR/Cas9 system introduces targeted double strand breaks in the genome, and the cells repair the break by error prone non-homologous end joining or homologous recombination resulting in insertion or deletion mutations. In this study, a CRISPR/Cas9 based system optimized for creating stable targeted gene knockouts in the marine diatom P. tricornutum was used to develop mutants for the LPA2 and LPA3 genes. From the screening of the transformed P. tricornutum colonies, one LPA2 knockout line was confirmed. Due to time limitations, the screening of all the transformed colonies was not completed. A high-resolution melt analysis indicated mutants in both the LPA2 and LPA3 transformed lines, however it was not confirm whether or not it was knockout mutations. The photosynthetic efficiency and cell division rate of two P. tricornutum LPA2 mutants (one heterozygote mutant and one homozygote knockout mutant), generated with two different gRNAs were examined in a HL experiment. These preliminary analyses indicate that the knockout mutation of the LPA2 gene leads to a decrease in the efficiency of the photosynthesis, and that LPA2 is possible involved in PSII assembly. To decide whether the disruption of the LPA2 gene in diatoms causes a truncated antenna, it would be necessary with further analyses on LPA2 knockout mutants. However, due to the normal coloration of the knockout mutant there is a low probability that it has a truncated antenna. Generation and characterization of knockout lines of LPA2 is therefore likely to contribute to the understanding of assembly of PSII, but it will probably not contribute to strain improvements useful for biotechnological applications as strain improvements on TLA mutants.