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De novo heterozygous variants in the brain-specific transcription factor Neuronal Differentiation Factor 2 (NEUROD2) have been recently associated with early-onset epileptic encephalopathy and developmental delay. Here, we report an adolescent with developmental delay without seizures who was found to have a novel de novo heterozygous NEUROD2 missense variant, p.(Leu163Pro). Functional testing using an in vivo assay of neuronal differentiation in Xenopus laevis tadpoles demonstrated that the patient variant of NEUROD2 displays minimal protein activity, strongly suggesting a loss of function effect. In contrast, a second rare NEUROD2 variant, p.(Ala235Thr), identified in an adolescent with developmental delay but lacking parental studies for inheritance, showed normal in vivo NEUROD2 activity. We thus provide clinical, genetic, and functional evidence that NEUROD2 variants can lead to developmental delay without accompanying early-onset seizures, and demonstrate how functional testing can complement genetic data when determining variant pathogenicity.

Cpf1 is a novel class of CRISPR-Cas DNA endonucleases, with a wide range of activity across different eukaryotic systems. Yet, the underlying determinants of this variability are poorly understood. Here, we demonstrate that LbCpf1, but not AsCpf1, ribonucleoprotein complexes allow efficient mutagenesis in zebrafish and Xenopus. We show that temperature modulates Cpf1 activity by controlling its ability to access genomic DNA. This effect is stronger on AsCpf1, explaining its lower efficiency in ectothermic organisms. We capitalize on this property to show that temporal control of the temperature allows post-translational modulation of Cpf1-mediated genome editing. Finally, we determine that LbCpf1 significantly increases homology-directed repair in zebrafish, improving current approaches for targeted DNA integration in the genome. Together, we provide a molecular understanding of Cpf1 activity in vivo and establish Cpf1 as an efficient and inducible genome engineering tool across ectothermic species., Cpf1 is a promising addition to the CRISPR toolkit but displays wide variability of activity in different eurkaryotes. Here the authors identify temperature as a modulator of activity and use this to efficiently edit ectothermic vertebrate species.

Mucociliary flow is an important defense mechanism in the lung to remove inhaled pathogens and pollutants. Disruption of ciliary flow can lead to respiratory infections. Multiple factors, from drugs to disease can cause an alteration in ciliary flow. However, less attention has been given to injury of the ciliated epithelium. In this study, we show how optical coherence tomography (OCT) can be used to investigate injury to the ciliated epithelium in a multi-contrast setting. We used particle tracking velocimetry (PTV-OCT) to investigate the cilia-driven flow field and 3D speckle variance imaging to investigate size and extent of injury caused to the skin of Xenopus embryos. Two types of injuries are investigated, focal injury caused by mechanical damage and diffuse injury by a calcium chloride shock. We additionally investigate injury and regeneration of cilia to calcium chloride on ex vivo mouse trachea. This work describes how OCT can be used as a tool to investigate injury and regeneration in ciliated epithelium.

CRISPR-Cas9 technology provides a powerful system for genome engineering. However, variable activity across different single guide RNAs (sgRNAs) remains a significant limitation. We analyzed the molecular features that influence sgRNA stability, activity and loading into Cas9 in vivo. We observed that guanine enrichment and adenine depletion increased sgRNA stability and activity, whereas differential sgRNA loading, nucleosome positioning and Cas9 off-target binding were not major determinants. We also identified sgRNAs truncated by one or two nucleotides and containing 5' mismatches as efficient alternatives to canonical sgRNAs. On the basis of these results, we created a predictive sgRNA-scoring algorithm, CRISPRscan, that effectively captures the sequence features affecting the activity of CRISPR-Cas9 in vivo. Finally, we show that targeting Cas9 to the germ line using a Cas9-nanos 3' UTR led to the generation of maternal-zygotic mutants, as well as increased viability and decreased somatic mutations. These results identify determinants that influence Cas9 activity and provide a framework for the design of highly efficient sgRNAs for genome targeting in vivo.

For the nematode Caenorhabditis elegans, automated selection of animals of specific genotypes from a mixed pool has become essential for genetic interaction or chemical screens. To date, such selection has been accomplished using specialized instruments. However, access to such dedicated equipment is not common. Here we describe live animal fluorescence-activated cell sorting (laFACS), a protocol for automatic selection of live first larval stage (L1) animals using a standard FACS system. We show that FACS can be used for the precise identification of GFP-expressing and non-GFP-expressing subpopulations and can accomplish high-speed sorting of live animals. We have routinely collected 100,000 or more homozygotes from a mixed starting population within 2 h, and with greater than 99% purity. The sorted animals continue to develop normally, making this protocol ideally suited for the isolation of terminal mutants for use in genetic interaction or chemical genetic screens.

The long bones of the vertebrate body are built by the initial formation of a cartilage template that is later replaced by mineralized bone. The proliferation and maturation of the skeletal precursor cells (chondrocytes) within the cartilage template and their replacement by bone is a highly coordinated process which, if misregulated, can lead to a number of defects including dwarfism and other skeletal deformities. This is exemplified by the fact that abnormal bone development is one of the most common types of human birth defects. Yet, many of the factors that initiate and regulate chondrocyte maturation are not known. We identified a recessive dwarf mouse mutant (pug) from an N-ethyl-N-nitrosourea (ENU) mutagenesis screen. pug mutant skeletal elements are patterned normally during development, but display a ~20% length reduction compared to wild-type embryos. We show that the pug mutation does not lead to changes in chondrocyte proliferation but instead promotes premature maturation and early ossification, which ultimately leads to disproportionate dwarfism. Using sequence capture and high-throughput sequencing, we identified a missense mutation in the Xylosyltransferase 1 (Xylt1) gene in pug mutants. Xylosyltransferases catalyze the initial step in glycosaminoglycan (GAG) chain addition to proteoglycan core proteins, and these modifications are essential for normal proteoglycan function. We show that the pug mutation disrupts Xylt1 activity and subcellular localization, leading to a reduction in GAG chains in pug mutants. The pug mutant serves as a novel model for mammalian dwarfism and identifies a key role for proteoglycan modification in the initiation of chondrocyte maturation.

We developed ultra-high-speed, phase-sensitive, full-field reflection interferometric confocal microscopy (FFICM) for the quantitative characterization of in vivo microscale biological motions and flows. We demonstrated 2D frame rates in excess of 1 kHz and pixel throughput rates up to 125 MHz. These fast FFICM frame rates were enabled by the use of a low spatial coherence, high-power laser source. Specifically, we used a dense vertical cavity surface emitting laser (VCSEL) array that synthesized low spatial coherence light through a large number of narrowband, mutually-incoherent emitters. Off-axis interferometry enabled single-shot acquisition of the complex-valued interferometric signal. We characterized the system performance (~2 μm lateral resolution, ~8 μm axial gating depth) with a well-known target. We also demonstrated the use of this highly parallelized confocal microscopy platform for visualization and quantification of cilia-driven surface flows and cilia beat frequency in an important animal model (Xenopus embryos) with >1 kHz frame rate. Such frame rates are needed to see large changes in local flow velocity over small distance (high shear flow), in this case, local flow around a single ciliated cell. More generally, our results are an important demonstration of low-spatial coherence, high-power lasers in high-performance, quantitative biomedical imaging.

A New Look at Old Data Although the full genome of Caenorhabditis elegans has been available for over 10 years, only a portion of the full-length messenger RNAs (mRNAs) and their expression through development has been analyzed. By combining multiple methods, involving a careful reannotation of existing data sets, sequencing of poly-A captured RNAs from multiple developmental stages, as well as cloning and deep sequencing of a subset of genes, Mangone et al. (p. 432 , published online 3 June) defined ∼26,000 distinct mRNA three-prime untranslated regions (3′UTRs) for ∼85% of the protein-coding genes. Most C. elegans genes have more than one 3′UTR, arising through different mechanisms including trans-splicing–coupled cleavage and polyadenylation. The work highlights the importance of the 3'UTRs in the regulation of mRNA stability and translation.

To the Editor: A recent paper in Nature Methods describes the use of fluorescence-activated cell sorting (FACS) is to sort Caenorhabditis elegans embryos1. Here we report FACS-based method to sort live C. elegans larvae, which permits us to rapidly collect large quantities of live genotyped worms from a mixed population. Using GFP-marked balancer chromosomes (Fig. 1a), and live-animal FACS laFACS, we routinely collected >100,000 genotyped animals in less than one hour. To test laFACS, we combined it with large-scale RNA interference (RNAi) screening and identified genetic interactors of mel-28, an important regulator of nuclear envelope and chromatin functions2–5. Figure 1 laFACS of GFP-negative L1 larvae. (a) Genetic scheme. A mel-28 mutant allele is kept over a balancer chromosome containing a GFP marker and a recessive lethal allele. GFP-negative progeny (F1) are mel-28 homozygotes and grow up to produce only dead embryos. ... Although a FACS machine is designed to sort single cells, a few modifications enabled sorting of ~0.25 mm long L1 C. elegans. First, we used a reduced drop-drive frequency of ~16.4 kHz to keep the larvae undamaged. We also used a 100-µm nozzle and set a gate to capture events with a high forward-scatter signal (Fig. 1b), confining our collections to larger objects (Supplementary Methods). After worm sorting, the same FACS machine could be used for several other applications, including sorting of yeast, Drosophila melanogaster cells, mammalian cells, and plant protoplasts. The worm sort had no impact on these applications, and the worm-specific FACS modifications were easily reversed to accommodate single-cell applications. We used laFACS to collect mel-28 (maternal-effect-lethal-28) homozygous worms from a mixed population. mel-28 homozygous hermaphrodites derived from heterozygous mothers appear phenotypically indistinguishable from wild-type animals until they mature and produce only inviable progeny. This selection is usually performed manually and thus is not amenable to large-scale applications. We first generated a GFP-marked strain in which the mel-28(t1684) mutation is balanced over a chromosome bearing lag-2::GFP6 (Fig. 1a). We sorted L1 worms, collecting GFP-negative mel-28(t1684) homozygotes. We selected GFP-negative larvae on the basis of the ratio of green (GFP fluorescence; 530/30 nm) to red (red-spectrum autofluorescence; 610/20 nm) signal (Fig. 1c,d). We separated GFP-positive and GFP-negative worms (Supplementary Figure 1). Typically, from a population of 600,000 larvae we retrieved ~130,000 healthy animals after one sort. This first-pass population was ~95-98% homozygous (n > 1,000, verified by microscopy). A second sort recovered an essentially 100% pure population of about 100,000 homozygous larvae (n > 20,000, verified by microscopy and by genetic analysis). This scheme can be easily adapted for the majority of C. elegans genes using available balancers. We used the collected homozygous mel-28 animals to perform an RNAi-based synthetic interaction screen using clones representing chromosome I genes7. The ability to easily collect large amounts of mel-28 homozygous animals allowed us to perform the RNAi repeatedly (up to 16 times). From over 2,000 genes tested, 12 showed synthetic phenotypes with mel-28: npp-2, npp-4, npp-12, npp-14, npp-17, his-67, his-68, exos-3, pas-5, phi-56, rpa-0, and rpl-30 (Supplementary Fig. 2 and Supplementary Table 1). These results agreed well with mel-28’s role in coordinating chromatin and nuclear envelope functions2,3. Obtaining large quantities of pure mutant populations could also be useful for chemical screens, microarrays, or biochemical assays, expanding the arsenal of high-throughput tools available in C. elegans.