CRISPR/Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We have analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observe that guanine enrichment and adenine depletion increase sgRNA stability and activity, while loading, nucleosome positioning and Cas9 off-target binding are not major determinants. We additionally identified truncated and 5′ mismatch-containing sgRNAs as efficient alternatives to canonical sgRNAs. Based on these results, we created a predictive sgRNA-scoring algorithm (CRISPRscan.org) that effectively captures the sequence features affecting Cas9/sgRNA activity in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos-3′-UTR fusion can generate maternal-zygotic mutants, increase viability and reduce somatic mutations. Together, these results provide novel insights into the determinants that influence Cas9 activity and a framework to identify highly efficient sgRNAs for genome targeting in vivo.

Identification of the coding elements in the genome is a fundamental step to understanding the building blocks of living systems. Short peptides (< 100 aa) have emerged as important regulators of development and physiology, but their identification has been limited by their size. We have leveraged the periodicity of ribosome movement on the mRNA to define actively translated ORFs by ribosome footprinting. This approach identifies several hundred translated small ORFs in zebrafish and human. Computational prediction of small ORFs from codon conservation patterns corroborates and extends these findings and identifies conserved sequences in zebrafish and human, suggesting functional peptide products (micropeptides). These results identify micropeptide-encoding genes in vertebrates, providing an entry point to define their function in vivo.

Cellular transitions require dramatic changes in gene expression that are supported by regulated mRNA decay and new transcription. The maternal-to-zygotic transition is a conserved developmental progression during which thousands of maternal mRNAs are cleared by post-transcriptional mechanisms. Although some maternal mRNAs are targeted for degradation by microRNAs, this pathway does not fully explain mRNA clearance. We investigated how codon identity and translation affect mRNA stability during development and homeostasis. We show that the codon triplet contains translation-dependent regulatory information that influences transcript decay. Codon composition shapes maternal mRNA clearance during the maternal-to-zygotic transition in zebrafish, Xenopus, mouse, and Drosophila, and gene expression during homeostasis across human tissues. Some synonymous codons show consistent stabilizing or destabilizing effects, suggesting that amino acid composition influences mRNA stability. Codon composition affects both polyadenylation status and translation efficiency. Thus, the ribosome interprets two codes within the mRNA: the genetic code which specifies the amino acid sequence and a conserved "codon optimality code" that shapes mRNA stability and translation efficiency across vertebrates.

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBa as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up-and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparb/d and the peroxisome proliferator-activated receptor a (PPARa) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.[Keywords: Circadian; miRNA; miR-122; metabolism; clock; PPAR] Supplemental material is available at http://www.genesdev.org.

MicroRNAs, or miRNAs, post-transcriptionally repress the expression of protein-coding genes. The human genome encodes over 1000 miRNA genes that collectively target the majority of messenger RNAs (mRNAs). Base pairing of the so-called miRNA ‘seed’ region with mRNAs identifies many thousands of putative targets. Evaluating the strength of the resulting mRNA repression remains challenging, but is essential for a biologically informative ranking of potential miRNA targets. To address these challenges, predictors may use thermodynamic, evolutionary, probabilistic or sequence-based features. We developed an open-source software library, miRmap, which for the first time comprehensively covers all four approaches using 11 predictor features, 3 of which are novel. This allowed us to examine feature correlations and to compare their predictive power in an unbiased way using high-throughput experimental data from immunopurification, transcriptomics, proteomics and polysome fractionation experiments. Overall, target site accessibility appears to be the most predictive feature. Our novel feature based on PhyloP, which evaluates the significance of negative selection, is the best performing predictor in the evolutionary category. We combined all the features into an integrated model that almost doubles the predictive power of TargetScan. miRmap is freely available from http://cegg.unige.ch/mirmap.