Journal: bioRxiv

Article Title: The Amyloid Precursor-like Protein APL-1 Regulates Axon Regeneration

doi: 10.1101/305284

Figure Lengend Snippet: The cytoplasmic C-terminus of endogenous APL-1 is not required for the inhibition of axon regeneration in GABAergic motor neurons. (A) Diagrams showing CRISPR/Cas9 mediated gene editing of the endogenous apl-1 locus, and the resulting protein produced. (SP signal peptide, AcD acidic domain, ICD intracellular domain, TM transmembrane domain, ΔC cytoplasmic c-terminal deletion) (B) Axon regeneration in apl-1ΔC mutant (wp22) is slightly decreased compared to juIs76 control. (Kolmogorov-Smirnov D=0.1684; P=0.0309) (C) Western blot comparing 3xFlag tagged endogenous APL-1 protein level in control, apl-1ΔC mutant, and rab-6.2(ok2254) mutant. Arrow indicates full length 3xFlag-APL-1 band. 3xFlag-APL-lΔC migrates at a slightly smaller size. Arrow head indicates actin (loading control). Quantification on the right shows four independent experiments (two lanes of rab-6.2 mutant were loaded for one of the experiments therefore 5 data points total). One way ANOVA and Tukey’s multiple comparisons test is used. Column and error bar shows mean with SEM. 3xFlag-APL-lΔC protein level is increased compared to 3xFlag-APL-1 control (Mean Difference=0.5425; P=0.0024). 3xFlag-APL-1 protein level is decreased in rab-6.2(ok2254) background compared to control (Mean Difference=-0.5989; P=0.0008).

Article Snippet: The Plasmids expressing Cas9 were obtained from Addgene #46168 ( ) and #47549 pDD162 ( ) (p46168 was used initially but we later switched to pDD162 when creating wp41 and wp42 , which gave us higher edit efficiency).

Techniques: Inhibition, CRISPR, Produced, Mutagenesis, Control, Western Blot

Journal: eLife

Article Title: A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen in Caenorhabditis elegans

doi: 10.7554/eLife.57874

Figure Lengend Snippet:

Article Snippet: recombinant DNA reagent , pDD162 (plasmid) , , Addgene plasmid #47549 , eft-3p::Cas9 + empty sgRNA plasmid.

Techniques: Migration, Homologous Recombination, Expressing, Membrane, Marker, Recombinant, Plasmid Preparation, Cloning, Generated, Clone Assay, Sequencing, Software

Journal: eLife

Article Title: Stem cell niche exit in C. elegans via orientation and segregation of daughter cells by a cryptic cell outside the niche

doi: 10.7554/eLife.56383

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , eft-3 p::Cas9+sgRNA expression vector , doi: 10.1534/genetics.115.178335 , RRID: Addgene_47549 , pDD162, Addgene plasmid #47549.

Techniques: Sequencing, Transgenic Assay, Knock-In, Mutagenesis, Recombinant, Modification, Plasmid Preparation, CRISPR, Expressing, Software

Journal: Nucleic Acids Research

Article Title: Highly efficient RNAi and Cas9-based auto-cloning systems for C. elegans research

doi: 10.1093/nar/gky516

Figure Lengend Snippet: The new C. elegans Cas9 vector Ccas9LacZ, and the efficiency of the auto-cloning system in Ccas9LacZ. ( A ) The original C. elegans Cas9 vector (up) and the new redesigned vector (down). The original C. elegans Cas9 vector does not include any restriction enzyme cleavage site between the U6 promoter and gRNA sequence that would allow the spacer sequence to be efficiently modified. The redesigned vector contains two Cas9 and two typeIIs restriction enzyme recognition sites next to the U6 promoter and gRNA sequences which allow the spacer sequence to be easily modified with restriction digestion coupled linker ligation and homologous recombination-based cloning too. The new vector also contains a lacZ gene that allows blue-white selection after cloning. ( B ) Sequencing of two white colonies chosen by enzyme digestion. The upper clone was made by the auto-cloning system, the clone at the lower part was made with restriction digestion coupled linker ligation, and both resulted the same sequence analogous to the predicted one. ( C ) Efficiency of Cas9 cloning was measured by colony number analysis. In case of restriction digestion-coupled linker ligation, in one experiment (Vector+AarI) we transformed only Ccas9LacZ vector digested with AarI enzyme to normal DH5alpha competent cells, in another one (Vector + AarI + Insert) we transformed both digested Ccas9LacZ vector and linker with appropriate overhangs. In case of auto-cloning system, in one experiment we transformed only Ccas9LacZ vector to 11 014 vector-containing competent cells (Vector + Cas9), in another one we transformed both Ccas9LacZ vector and spacer sequence-containing inserts with three different homology arm length of 25, 50 and 100 bp (Vector + Cas9 + 25 bp, Vector + Cas9 + 50 bp and Vector + Cas9 + 100 bp). Blue bars indicate colonies containing non-cleaved, original T777T vectors, white bars indicate colonies with plasmids lacking the insert sequence, and bars marked with green are colonies with vectors containing the insert. Bars represent ±S.D.

Article Snippet: Ccas9LacZ was generated from the pDD162 (Peft-3::Cas9 + Empty sgRNA, Addgene no.: #47549) and T777T.

Techniques: Plasmid Preparation, Cloning, Sequencing, Modification, Ligation, Homologous Recombination, Selection, Transformation Assay