Cells sense their environment and adapt to it by fine-tuning their transcriptome. Wired into this network of gene expression control are mechanisms to compensate for gene dosage. The increasing use of reverse genetics in zebrafish, and other model systems, has revealed profound differences between the phenotypes caused by genetic mutations and those caused by gene knockdowns at many loci, an observation previously reported in mouse and Arabidopsis. To identify the reasons underlying the phenotypic differences between mutants and knockdowns, we generated mutations in zebrafish egfl7, an endothelial extracellular matrix gene of therapeutic interest, as well as in vegfaa. Here we show that egfl7 mutants do not show any obvious phenotypes while animals injected with egfl7 morpholino (morphants) exhibit severe vascular defects. We further observe that egfl7 mutants are less sensitive than their wild-type siblings to Egfl7 knockdown, arguing against residual protein function in the mutants or significant off-target effects of the morpholinos when used at a moderate dose. Comparing egfl7 mutant and morphant proteomes and transcriptomes, we identify a set of proteins and genes that are upregulated in mutants but not in morphants. Among them are extracellular matrix genes that can rescue egfl7 morphants, indicating that they could be compensating for the loss of Egfl7 function in the phenotypically wild-type egfl7 mutants. Moreover, egfl7 CRISPR interference, which obstructs transcript elongation and causes severe vascular defects, does not cause the upregulation of these genes. Similarly, vegfaa mutants but not morphants show an upregulation of vegfab. Taken together, these data reveal the activation of a compensatory network to buffer against deleterious mutations, which was not observed after translational or transcriptional knockdown.

Genetic compensation induced by deleterious mutations but not gene knockdowns

Fourth generation biofuel (FGB) uses genetically modified (GM) algae to enhance biofuel production. Although GM algae biofuel is a well-known alternative to fossil fuels, the potential environmental and health-related risks are still of great concern. An evaluation of these concerns and accordingly devising appropriate mitigation strategies to deal with them are important to a successful commercialized production of FGB. While extensive research has been carried out on genetic modification and other technologies that aim to increase the productivity of algae strains, only a handful of them deal with the legislative limitations imposed on exploiting and processing GM algae. This paper examines this legislation and the mitigation strategies to meet potential risks associated with the exploitation and processing of FGB. Open-pond system is an economic solution for large-scale cultivation of microalgae; however, the concern regarding the health and environmental risk of cultivating GM algae and the associated stringent regulations is considered as the main barrier of FGB production. Disposal of the residue is another important issue that should be considered in FGB production. The byproducts obtained from energy extraction step and residual water from the harvesting process may contain plasmid or chromosomal DNA that may cause the risk of lateral gene transfer. Hence an appropriate mitigation practices should be used for replacement of the hazardous water residue and by-products with more environmentally friendly alternatives. The results obtained from several field testing projects for open-environment exploitation of GM algae show that under the various conditions used, there was no apparent proof to support possible horizontal gene transfer in release of GM algae.

Fourth generation biofuel: a review on risks and mitigation strategies

Marine microalgae for production of biofuels and chemicals

Phaeodactylum tricornutum: A Diatom Cell Factory.

Recently developed transgenic techniques to explore and exploit the metabolic potential of microalgae present several drawbacks associated with the delivery of exogenous DNA into the cells and its subsequent integration at random sites within the genome Here, we report a highly efficient multiplex genome-editing method in the diatom Phaeodactylum tricornutum, relying on the biolistic delivery of CRISPR-Cas9 ribonucleoproteins coupled with the identification of two endogenous counter-selectable markers, PtUMPS and PtAPT First, we demonstrate the functionality of RNP delivery by positively selecting the disruption of each of these genes Then, we illustrate the potential of the approach for multiplexing by generating double-gene knock-out strains, with 65% to 100% efficiency, using RNPs targeting one of these markers and PtAureo1a, a photoreceptor-encoding gene Finally, we created triple knock-out strains in one step by delivering six RNP complexes into Phaeodactylum cells This approach could readily be applied to other hard-to-transfect organisms of biotechnological interest

/pdf/one-step-generation-of-multiple-gene-knock-outs-in-the-2dporp9vhx.pdf

One-step generation of multiple gene knock-outs in the diatom Phaeodactylum tricornutum by DNA-free genome editing.

Reverse genetics techniques are powerful tools for studying gene functions. In the model diatom Phaeodactylum tricornutum , RNAi-mediated knockdown of genes still is the most commonly used reverse genetics technique. Due to the diploidic life cycle missing reproduction in lab cultures, many commonly used techniques to create knockout instead of knockdown lines are not applicable in P. tricornutum . These limitations can be overcome by using genome editing approaches like TALEN (Transcription activator-like effector nucleases), and/or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats), allowing the introduction of targeted mutagenesis events. Both techniques have recently been adapted exemplarily for diatoms, however, no concise guidelines exist yet for routine utilization of these tools and the subsequent characterization of the mutants. We therefore have adapted a cost-effective TALEN generation system previously established for mammalian cells for the use in P. tricornutum, allowing the assembly of TALENs in about two weeks. We further provide protocols for: a) choosing a TALEN target site in order to avoid potentially ineffective and/or off-target prone TALEN constructs, b) efficient transformation of P. tricornutum with both TALEN constructs, utilizing two antibiotics resistance markers, c) effective screening of the transformants. In order to test our system we chose the blue-light dependent transcription factor Aureochrome 1a ( PtAureo1a ) as a target gene due to the known phenotype of previously characterized P. tricornutum RNAi knockdown strains. Our TALEN approach appears to be highly efficient: targeted mutation events were detected in 50% of all transformants obtained, whereas 21% of the transformants were found to be bi-allelic knockout lines. Furthermore, most TALEN transformed cell lines were found to be genetically homogeneous without the need for re-plating, which greatly facilitates the screening process.

/pdf/a-fast-and-reliable-strategy-to-generate-talen-mediated-gene-2hcxq41m5u.pdf

A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum

The recent availability of genome editing tools like TALEN (Transcription activator-like effector nuclease) and CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats) for the diatom Phaeodactylum tricornutum has dramatically increased the options to explore diatom biology via reverse genetics. In order to verify that an observed phenotype indeed is directly related to a specific gene knockout and not due to a secondary effect, complementation of the inactivated gene with the wildtype gene and restoration of the wild type phenotype is an essential tool in molecular biology. So far, no strategy for a complementation method has been published for P. tricornutum. Here we demonstrate, as a proof-of principle, the complementation of P. tricornutum AUREO1a knockout strains previously created by TALEN technology. These strains are deficient in the PtAureo1a gene, which is encoding a blue-light dependent transcription factor. pPTbsr, a modified pPha-T1 vector with an antibiotic resistance cassette against Blasticidin served as a complementation vector. In order to avoid the modification of the complementing gene via the potentially still active TALEN nucleases, we have modified the TALEN binding sites of the complementing PtAureo1a gene using synonymous codons. The altered PtAureo1a gene along with its native promoter and terminator was transformed by particle gun bombardment into PtAUREO1a TALEN knockout strains of P. tricornutum. The integration and the expression of PtAUREO1a was confirmed by PCR and western blotting. Due to random integration within the genome, the expression level of the complemented gene may be variable in different lines. Physiological parameters indicated the successful rescue of the wild type phenotype in several lines that showed a similar PtAUREO1a protein content as wild type cells. Our method provides a rapid and efficient tool to complement knockout lines generated by genome editing approaches in P. tricornutum.

C. Rio Bartulos

Papers

A fast and reliable strategy to generate TALEN-mediated gene knockouts in the diatom Phaeodactylum tricornutum

A strategy to complement PtAUREO1a in TALEN knockout strains of Phaeodactylum tricornutum