The Chin-Sang Lab Reads

Papers selected January- 2015

C. elegans L1 straved animals  can metaboize EtOH to induced a aggregration behaviour.

Auxin induced protein degradation in C. elegans.

 rab-35, rab-10 and M04F3.2,  as well as vrp-1, ceh-60 and lrp-2  are need for abudant yolk protein.  Surprisingly, worms do fine with reduced yolk proteins.

Differential regulation of germline stem cell proliferation rates in C. elegans adults is accomplished through localized inhibition of insulin/IGF-1 signalling, requiring daf-18/PTEN, but not daf-16/FOXO. 

Review on PTEN Regualton

Starving C. elegans at L1 take longer to become reproductive, have lower fecundity and were smaller as adults. The progeny and grandprogeny of starved L1 animals were more resistant to starvation and heat stress. 

Paix et al. use synthesized tracrRNA and crRNAs and recobinant  Cas9 to inject into C. elegans. Supp. material.

We have tried this protocol and it works really well. E. coli will take linearized vector and insert with 20bp homology overlaps  (like Gibson assembly but without enzyme mix) and recombine the two pieces into a circular plasmid. We have tried with DH5 alpha, Top10 and XL1Blue.  We treat with  T4 DNA polymerase (3’ Exonuclease activity)  for 2.5 minutes  and this increases the efficiency.  See this SLIC paper and protocol.  Here is another paper that uses 40-50bp homolgy overlap and can recombine multiple fragments. And another one calle AQUA cloning. They also use this method for insertions, deletions, or substitutions. The method shows a broad compatibility with most widely used lab-strains of E. coli, including TOP10, NEB5α, NEB10β, BL21 (DE3) and JM109. 

Dickinson et al. revised their method for Crispr genome engineering in C. elegans. Improvements include drug selection and a Cre mediated self excision leaving only a small scar within an intron. In a single micro-injection they could isolate, 1) knock out for his-72 2) a his-72 transcriptional reporter and 3) a HIS-72 translational reporter. Detailed Protocol

Sense oligo for HR seems to work better than anti-sense and homology independent DNA repair  (like in zebrafish) works in C. elegans.

The sense oligo could be explained by this paper:

1) After duplex cleavage, Cas9 holds onto three ends of the target DNA (white crossed circles), but the PAM-distal non-target strand is released from the Cas9-DNA complex. 2) Complementary DNA anneals to released strand. 3) Branch migration results in extrusion from the Cas9-DNA complex.

Note that the pha-1 guide RNA targets the sense strand and thus the Oligo for repair does conform to this rule 

A new model, in addition to lagging strand hypothesis, to explain Gap repair in lambda Red recombineering.

The authors (Canadian) show the Crispr/Cas9 cleavage coupled to lambda Red recombineering can create large gene edits eg. deletions (94.3kb) or insertions  (3kb). Without any selection (just colony PCR).

Andrew Chisholm’s review on wound healing in C. elegans.

The authors report that they get higher efficiency of HDR  reporter knock-in mice by using  dual-crRNA:tracrRNA combined with Cas9 protein and repair template. A similar approach has been reported in zebrafish and C. elegans.

 A review on Eph–ephrin signaling in adhesion, repulsion and tension—that can in principle underlie the segregation of cells and formation of sharp borders. 

Recent experiments led to insights into how the widely used laboratory reference strain of the nematode Caenorhabditis elegans compares with natural strains. 

The natural history of C. elegans.

Methods Volumes 77–78, Pages 1-204 (1 May 2015) PTEN Function Methods

Designing your guide RNA to have GG just before the PAM site increases CRISPR/Cas9 edits.

Ploymerase theta is used in the C. elegans germline to to promote end joining after double strand break.  The authors show that  CRISPR/Cas9-induced genomic changes are exclusively generated through polymerase theta-mediated end joining, refuting a previously assumed requirement for NHEJ in their formation. 

Reduced insulin can increase longevity through a dauer independent pathway through SKN-1 (Nrf, NF-E2-related factor)which increases expression of collagens and other extracellular matrix genes.

 We tend to forget to understand how the pioneer researchers faced major questions. 

Lysomes have a signaling role in aging.

A review on transgenerational epigenetics.

Quality vs Quantity of life is addressed in C. elegans.

The E3 ubiquitin ligase substrate-recognition subunit ZIF-1, can be used to proteins and  can be quickly degraded in all somatic cell types examined with temporal and spatial control.

Review on monitoring autophagy in C. elgans.