Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii.

In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (HR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR. This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.

Knock-Down of a ligIV Homologue Enables DNA Integration via Homologous Recombination in the Marine Diatom Phaeodactylum tricornutum.

Genetic engineering of Phaeodactylum tricornutum as a model organism for diatoms is the basis of molecular and biochemical research, and can also be used in biotechnological approaches. So far, integration of foreign DNA into the genome happens randomly by nonhomologous end joining (NHEJ), if the classical method of particle bombardment is used, with the danger of negative physiological side effects. Here we show that a putative gene for a DNA ligase IV homologue ( ligIV) in P. tricornutum codes for a functional LigIV. The knock-down of ligIV in P. tricornutum via antisense RNA drastically enhances homologous recombination (HR) by interfering with the NHEJ pathway at its central DNA ligation step done by LigIV. This enables a specific integration of DNA at desired locations, greatly enhanced transformation rates and provides a new way of specifically altering the genome of P. tricornutum.

Identification of a triacylglycerol lipase in the diatom Phaeodactylum tricornutum.

Diatoms accumulate triacylglycerols (TAGs) as storage lipids, but the knowledge about the molecular mechanisms of lipid metabolism is still sparse. Starting from a partial sequence for a putative TAG-lipase of the diatom Phaeodactylum tricornutum retrieved from the data bases, we have identified the full length coding sequence, tgl1. The gene encodes an 813 amino acid sequence that shows distinct motifs for so called "true" TAG-lipases [EC 3.1.1.3] that have been functionally characterized in model organisms like Arabidopsis thaliana and Saccharomyces cerevisiae. These lipases mediate the first initial step of TAG breakdown from storage lipids. To test whether Tgl1 can act as a TAG-lipase, a His-tagged version was overexpressed in Escherichia coli and the protein indeed showed esterase activity. To identify the TAG degrading function of Tgl1 in P. tricornutum, knock-down mutant strains were created using an antisense RNA approach. In the mutant cell lines the relative tgl1-mRNA-level was reduced up to 20% of that of the wild type, accompanied by a strong increase of TAG in the lipid extracts. In spite of the TAG accumulation, the polar lipid species pattern appeared to be unchanged, confirming the TAG-lipase function of Tgl1.