The Chin-Sang Lab reads

Papers selected January -December 2014

Jordan Ward has provided new improvements for better efficiency of CRISPR Cas9 HDR. 1) Use a better sgRNA (sgRNA F+E) 2) Use the conversion of pha-1(ts) as a selection and score these animals for conversion (co-conversion strategy) 3) Use ssODN as repair template 4) Inhibit NHEJ  (cku-80 RNAi). Update: At the last worm meeting they mentioned that inhibiting NHEJ doesn’t seem to help that much.

A nice history of the discovery of  Crispr Cas9 

Drosophila Eph RTK signaling required for compartment boundary formation.

 Optimization of CRISPR/Cas9 in fly shows that sgRNAs with high GC content before PAM and 18 nt seed vs. 20 nt is more efficient. 

Starvation in C. elegans induces transgenerational inheritance of small RNAs

Even without histone methyl transferase (PRC2), H3K27me is transmitted to daughter chromatids after ell division. In embryos with PRC2, H3K27me is only seen on chromosomes that had had  existing marks. See this preview.

Epidermal wounding triggers local production of the mitochondrial ROS (mtROS) superoxide and promotes actin mediated wound repair. Ca2+ uptake and inhibition of Rho activity is needed at wound site.

Review on the methods to study  PTEN subcellular localization

Chemosensation of  henazine-1-carboxamide and pyochelin produced by Pseudomonas aeruginosa activate a G-protein-signaling pathway that induces expression of the TGF-β and promotes avoidance behavior.

PHA-4, and SKN-1 regulate miR-71 and miR-228 are critical for dietary-restriction-induced longevity. PHA-4 and SKN-1 are negatively regulated by miR-228

Here Paix et al. report that CRISPR/Cas9  HDR can be done efficiently using just 30-60bp of homology arms.  Previously other groups would use about 1kb for homology arms. Detailed Protocol here.

A review on the molecular genetics of decision making.

ins-4, daf-28 and to a lesser extent ins-6 function redundantly  to  regulate L1 arrest.

 Sema1a reverse signaling promotes the phosphorylation and activation of Moesin (Moe), a member of the ezrin/radixin/moesin family of proteins, and downregulates the level of active Rho1 in photoreceptor axons. 

Grizzly bears prepare for hibernation by increased insulin sensitivity via down regulation of PTEN in fat tissue.

a three-dimensional scaffold of heparan sulfate mediates KAL-1/anosmin-1 

 insulin signaling strength can differentially affect specific neurons aging naturally or under proteotoxic stress

DAF-16/FOXO, and  DAF-18/PTEN and several components of the proteasome complex (rpn-6.1, rpn-7, rpn-9, rpn-10, rpt-6, pbs-3 and pbs-6) are essential for both lifespan and immunity of germline deficient animals. 

A review on the  mechanosensory systems of C. elegans, including the sensory neurons and circuitry involved in body touch, nose touch, and proprioception.

In the Q neuroblast lineage, loss of GRP- 1 (cytohesin)  led to the production of daughter cells that are more similar in size and the cells that should undergo apoptosis survives , resulting in the production of extra neurons.

The BK channel T352I missense mutation was insensitive to activation by ethanol.

Expression of vertebrate connexins in C. elegans  can be used to synthetically connect neurons.

A review on C. elegans approaches to target protein folding diseases.

ANI-2 promotes germ cell syncytial organization and allows for compensation of the mechanical stress associated with oogenesis by conferring stability and elasticity to germ cell intercellular bridges.

Review on the FMRF-amide neuropeptides.

Knocking out genes by feeding CRISPR guide RNAs.

During the oocyte to Embryo transition, many maternal mRNAs are cleared. A polyC motif in the 3′ UTR serves as a target for polyC binding proteins likely initiated by endogenous siRNAs.

The Wnt receptor Ryk, is increased in different animal models of HD. In C. elegans lin-18/Ryk represses daf-16/FOXO protective activity, possibly directly, through its intracellular domain, a product of γ-secretase–mediated cleavage previously implicated in the birth of new cortical neurons. 

Normally Q.aa and Q.pp are smaller cells that undergo apoptosis. The DEP domain-containing protein TOE-2 in promotes the apoptotic fate in the Q lineage. Interestingly in toe-2 mutants the Q.aa was a similar size to its sister but int he Q.pp daughter was smaller but did not undergo apoptosis.   Also see the PIG-1 paper1. and paper 2 

Metformin is able to extend lifespan by increasing the production of reactive oxygen species 

Some of the physiological phenotypes previously attributed to peripheral AAK-2 activity on metabolic targets may instead be due to the role of this kinase in neural serotonin signaling.

When C. elegans  L3 or L4 worms are removed form food development stops (eg. vulva formation).  DAF-16, a major target of insulin-like signaling, functions to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormone.

The UNC-6/Netrin guidance cue has a conserved role in guiding cell and growth cone migrations along the dorso-ventral axis, whereas Wnts are critical for determining polarity and guidance along the antero-posterior axis. In this study we show that these two signaling pathways function redundantly in both antero-posterior and dorso-ventral guidance 

 The authors have made an enhanced PTEN (ePTEN) with approximately eightfold increased ability to suppress PIP3 signaling. This involved making 5 mutations in the PTEN protein: ePTEN Q17R, R41G, E73D, N262Y, N329H.

Deletion of TRPV1, an ion channel critical for nociception, extends mouse and C. elegans lifespan by regulating the activity of CRTC1 in peripheral sensory neurons.

 I motor neurons and mechanosensory neurons RPM-1 is localized to multiple, distinct subcellular compartments in a single cell to coordinate axon outgrowth and termination with synapse formation.

Actin polymerization and myosin II-mediated contractility may not be required for some cells in confined migration. Water permeation regulates cell volume and drives migration in narrow channels. 

Using a known to work sgRNA (unc-22) with your sgRNA you can let you know when the Cas9 activity has occurred. The authors also show you can directly select for GFP tagged genes by homologous recombination.

RPM-1 functions through a PP2C phosphatase, PPM-2, to regulate the activity of a MAP3 kinase, DLK-1. 

The authors use an In vivo sensor for mechanical tension to show differences in the front and back of migrating cells.

A web tool for identifying mutations from WGS.  Link to Mudi.  Also see CloudMap.

LIN-32 controls Q cell division and migrations.

 3 different imaging techniques that can be used to image live C. elegans without the use of microfluidic technologies

This paper presents a new type of nuclei segmentation method for automated analysis of cell lineage.

 Pheromones sensed by ciliated neurons in the Caenorhabditis elegans nose alter the lipid microenvironment within the oviduct, thereby affecting sperm motility. 

α-KG inhibits ATP synthase and,  and leads to reduced ATP content, decreased oxygen consumption, and increased autophagy in both C. elegans and mammalian cells.  This leads to extended lifespan.

WormNet v3 is freely accessible at

Deep sequencing strategies for simultaneously mapping and identifying causal mutations in Caenorhabditis elegans from mutagenesis screens. Have a look at CloudMap a cloud based pipeline for analysis of mutant genome sequences. 

Although CRISPR will likely replace Mos1 meditated genome editing there are two compelling uses for miniMos: 1) will be to probe the genome on a global scale for chromatin effects and to determine expression patterns using gene-trap constructs. 

Review of the CRISPR/Cas9 methods used in C. elegans.

daf-18 and shc-1;daf-18 mutants develop a disrupted gonad after L1 starvation. This is not dependent on the L1 arrest germline proliferation as daf-18(e1375) does not show proliferation at L1 arrest but nonetheless has a disrupted gonad.  hyper-proliferation and basement membrane disruption characterize this phenotype.  daf-2(lf) can suppress  proper gonad development in a DAF-16 independent manner.  Doxycyclin ( selectively inhibits mitochondrial protein synthesis) and reduced starvation temperature can suppress the phenotypic penetrance

Molecular mechanisms by which free radicals can have a pro-longevity effect. See below James Watson’s hypothesis on why causes type 2 diabetes.

Reduced insulin signaling restores germ cell immortality to prg-1.  This is accomplished by activating a parallel nuclear RNA-silencing pathway.

James Watson’s hypothesis of the cause of type 2 diabetes "“The fundamental cause, I suggest, is a lack of biological oxidants, not an excess,” he says. "Physical exercise prompts the body to make large numbers of oxidants — molecules called reactive oxygen species, or ROS," he continues, and that's why exercise is beneficial to our bodies.

Review on Axon regeneration in C. elegans.

EphA–Vav2–RhoA-mediated repulsion between contacting cancer cells at the tumour edge could enhance their local invasion away from the primary tumour.

PINK1-dependent phosphorylation of both parkin and ubiquitin is sufficient for full activation of parkin E3 activity.

Review on using C. elegans to model neurodegenerative diseases.

 PTEN alpha is longer isoform that uses a CUG start codon.PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. 

Review on the mechanisms that control PTEN localization.

PTEN forms a dimer for active PIP3 phosphatase activity. Some PTEN missense mutations found cancers act as dominant negative.

RNA-binding protein Musashi regulates forgetting in C. elegans through translational repression of the actin-branching Arp2/3 complex. Memory length is regulated cooperatively through the activation of Adducin and by the glutamate-receptor/Musashi-1/Arp2/3 pathway

An automated system simultaneously images worm behavior and neuronal Ca2+ transients.

 Genome editing in adult animals.  CRISPR-Cas9–mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. They used hydrodynamic tail vein injection to get DNA into the liver cells.

The forty insulin-like peptides in the nematode C. elegans have specific and distinct effects on eight different physiological outputs that range from development, stress responses, lifespan and reproduction. Interestingly, we also find that these peptides regulate each other at the transcriptional

A method for single/low copy integration of transgenes.

 Cam Kinase II (UNC-43)  has a role in blocking the regulated exocytosis of Dense Core Vesicles  before they are transported into axons.

Review on microfluidic platforms for C. elegans research.

 A review on Ephrin signaling in the developing nervous system.

 A  review of the use of CRISP/Cas9 in Drosophila. 

PNKP is synthetic lethal with PTEN.  Cells have increased apoptosis and increased radiosensitivity in the double knock down. The worm homolog of PNKP is  F21D5.5   

 A method  to silence neurons by expressing an histamine-gated chloride channel (HisCl1) in specific neurons. The activity of HisCl1-expressing neurons is acutely and reversibly silenced by exogenous histamine, which C. elegans absorbs from the media. 

Reprogramming cells by stimulus-triggered acquisition of pluripotency (STAP), which requires neither nuclear transfer nor the introduction of transcription factors.

Genetics screens  identified at least 50 conserved genes with growth-promoting or growth-inhibiting functions for axon regeneration in C. elegans.