CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass.

The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.

Elevated basal transcription can underlie timothy channel association with autism related disorders.

Timothy syndrome (TS) is a neurodevelopmental disorder caused by mutations in the pore-forming subunit α11.2 of the L-type voltage-gated Ca2+-channel Cav1.2, at positions G406R or G402S. Although both mutations cause cardiac arrhythmias, only Cav1.2G406R is associated with the autism-spectrum-disorder (ASD). We show that transcriptional activation by Cav1.2G406R and Cav1.2G402S is driven by membrane depolarization through the Ras/ERK/CREB pathway in a process called excitation-transcription (ET) coupling, as previously shown for wt Cav1.2. This process requires the presence of the intracellular ß-subunit of the channel. We found that only the autism-associated mutant Cav1.2G406R, as opposed to the non-autistic mutated channel Cav1.2G402S, exhibits a depolarization-independent CREB phosphorylation, and spontaneous transcription of cFos and MeCP2. A leftward voltage-shift typical of Cav1.2G406R activation, increases channel opening at subthreshold potentials, resulting in an enhanced channel activity, as opposed to a rightward shift in Cav1.2G402S. We suggest that the enhanced spontaneous Cav1.2G406R activity accounts for the increase in basal transcriptional activation. This uncontroled transcriptional activation may result in the manifestation of long-term dysregulations such as autism. Thus, gating changes provide a mechanistic framework for understanding the molecular events underlying the autistic phenomena caused by the G406R Timothy mutation. They might clarify whether a constitutive transcriptional activation accompanies other VGCC that exhibit a leftward voltage-shift of activation and are also associated with long-term cognitive disorders.

Molecular characterization and expression analysis of CSαß defensin genes from the scorpion Mesobuthus martensii.

Defensins are important components of innate host defence system against bacteria, fungi, parasites and viruses. Here, we predicted six potential defensin genes from the genome of the scorpion Mesobuthus martensii and then validated four genes from them via the combination of PCR and genomic sequence analysis. These four scorpion defensin genes share the same gene organization and structure of two exons and one phase-I intron with the GT-AG rule. Conserved motif and phylogenetic analysis showed that they belonged to the members of the invertebrate cysteine-stabilized α-helix/ß-sheet motif defensin (CSαß) defensin family. All these four CSαß defensin genes have the expression feature of constitutive transcription (CON) by the whole scorpion infection model, promoter sequence analysis and dual luciferase assays. Further evolution and comparison analysis found that the invertebrate CSαß defensin genes from most of arachnids and mollusks appear to share the expression pattern of CON, but those from insects and lower invertebrates (nematodes, annelids, cnidarians and sponges) seem to have identical inducible transcription (IND) after being challenged by microorganisms. Together, we identified four scorpion CSαß defensin genes with the expression feature of CON, and characterized the diversified expression patterns of the invertebrate CSαß defensin genes, which will shed insights into the evolution of the invertebrate CSαß defensin genes and their expression patterns.