ncOrtho: efficient and reliable identification of miRNA orthologs.

MicroRNAs (miRNAs) are post-transcriptional regulators that finetune gene expression via translational repression or degradation of their target mRNAs. Despite their functional relevance, frameworks for the scalable and accurate detection of miRNA orthologs are missing. Consequently, there is still no comprehensive picture of how miRNAs and their associated regulatory networks have evolved. Here we present ncOrtho, a synteny informed pipeline for the targeted search of miRNA orthologs in unannotated genome sequences. ncOrtho matches miRNA annotations from multi-tissue transcriptomes in precision, while scaling to the analysis of hundreds of custom-selected species. The presence-absence pattern of orthologs to 266 human miRNA families across 402 vertebrate species reveals four bursts of miRNA acquisition, of which the most recent event occurred in the last common ancestor of higher primates. miRNA families are rarely modified or lost, but notable exceptions for both events exist. miRNA co-ortholog numbers faithfully indicate lineage-specific whole genome duplications, and miRNAs are powerful markers for phylogenomic analyses. Their exceptionally low genetic diversity makes them suitable to resolve clades where the phylogenetic signal is blurred by incomplete lineage sorting of ancestral alleles. In summary, ncOrtho allows to routinely consider miRNAs in evolutionary analyses that were thus far reserved to protein-coding genes.

Minimized combinatorial CRISPR screens identify genetic interactions in autophagy.

Combinatorial CRISPR-Cas screens have advanced the mapping of genetic interactions, but their experimental scale limits the number of targetable gene combinations. Here, we describe 3Cs multiplexing, a rapid and scalable method to generate highly diverse and uniformly distributed combinatorial CRISPR libraries. We demonstrate that the library distribution skew is the critical determinant of its required screening coverage. By circumventing iterative cloning of PCR-amplified oligonucleotides, 3Cs multiplexing facilitates the generation of combinatorial CRISPR libraries with low distribution skews. We show that combinatorial 3Cs libraries can be screened with minimal coverages, reducing associated efforts and costs at least 10-fold. We apply a 3Cs multiplexing library targeting 12,736 autophagy gene combinations with 247,032 paired gRNAs in viability and reporter-based enrichment screens. In the viability screen, we identify, among others, the synthetic lethal WDR45B-PIK3R4 and the proliferation-enhancing ATG7-KEAP1 genetic interactions. In the reporter-based screen, we identify over 1,570 essential genetic interactions for autophagy flux, including interactions among paralogous genes, namely ATG2A-ATG2B, GABARAP-MAP1LC3B and GABARAP-GABARAPL2. However, we only observe few genetic interactions within paralogous gene families of more than two members, indicating functional compensation between them. This work establishes 3Cs multiplexing as a platform for genetic interaction screens at scale.

Thermoanaerobacter species differ in their potential to reduce organic acids to their corresponding alcohols.

The reduction of organic acids to their corresponding alcohols has been shown for some bacterial species within the Firmicutes super-phylum and a genetically modified strain of the hyperthermophilic archaeon Pyrococcus furiosus. In the latter strain, an aldehyde:ferredoxin oxidoreductase (AOR) catalyzed the reduction of a variety of organic acids to their corresponding aldehydes, as shown by the deletion of the corresponding aor gene. Here, we found that the genomes of a few thermophilic bacterial species within the genus Thermoanaerobacter which have been described to efficiently ferment sugars to ethanol harbor a copy of aor, while others do not. Specific AOR activity was only found in strains with aor, and the gene was highly expressed in Thermoanaerobacter sp. strain X514. The reduction of a variety of organic acids was observed for several Thermoanaerobacter sp.; however, strains with aor reduced, e.g., isobutyrate at much higher rates of up to 5.1 mM h-1 g-1. Organic acid reduction also led to increased growth rates in Thermoanaerobacter sp. strain X514 and in Thermoanaerobacter pseudethanolicus. Organic acid activation may proceed via acyl-CoA with subsequent NADH-dependent reduction by an aldehyde dehydrogenase (ALDH), or via direct reduction by AOR. Cell-free extracts of Thermoanaerobacter sp. strain X514 exhibited both enzyme activities at comparable rates. Therefore, the biochemistry of organic acid reduction to alcohols in Thermoanaerobacter sp. remains to be elucidated; however, relatively high specific activities and the correlation of AOR specific activities with alcohol production rates suggest a role for AOR.