Journal: bioRxiv

Article Title: Genetic exploration of a nuclear receptor transcriptional regulatory complex

doi: 10.1101/2020.03.28.013060

Figure Lengend Snippet: (A) lir-1 and lin-26 genes are located downstream of lir-2 . NHR-25 ChIP-seq data (Thurtle-Schmidt et al., unpublished data) revealed NHR-25 occupied peak found in the long intron of lir-1 at about 3 kb upstream of the lir-1 / lin-26 operon. (B) The NHR-25 ChIP-seq peak sequence is highly conserved with other Caenorhabditis species (Multiz alignment, UCSC Genome Browser) and two NR5A-recognition consensus are found (boxed in blue) by JASPAR2020 (scan against NR5A2 matrix ID MA0505.1). (C) depicts NHR-25 ChIP-seq peak (blue bar), NR5A recognition elements (NR5ARE) are shown (light orange bars). Dark orange bars indicate legions in CRISPR-Cas9 generated deletion mutants (jm199, jm200 strains). (D) Brood size analyses of NR5ARE deletion mutant strains (left), CRISPR-Cas9 generated lir-2 mutants and original strain HL178 (right) at 20°C. 5-8 animals for each strain were scored. Standard error of the mean (SEM) is shown for each bar.

Article Snippet: Briefly, CRISPR-Cas9 ribonucleoprotein (RNP) complexes were preassembled by mixing 110 ng/μL tracr RNA (IDT), 100 ng/μL of each guide RNAs (Alt-R crRNA, IDT) and 250 ng/μL Cas9-NLS purified protein (MacroLab, QB3 Berkeley) and incubating at 37 °C for 10 min. 110 ng/μL of each ssODN donor templates (IDT) and 50 ng/μL pRF4: co-injection marker rol-6 (su1006) expression plasmid ( Kramer et al. 1990 ) were then added to the complex mixture.

Techniques: ChIP-sequencing, Sequencing, CRISPR, Generated, Mutagenesis

Journal: bioRxiv

Article Title: Genetic exploration of a nuclear receptor transcriptional regulatory complex

doi: 10.1101/2020.03.28.013060

Figure Lengend Snippet: (A) lir-2 genomic and protein structures are shown. Mutation S9 is indicated in red asterisk. Three LxxLL motifs are found at the position 161, 211 and 290 in LIR-2 and marked in green. The region containing LxxLL motifs (Lx1, Lx2 and Lx3) were aligned with ogous proteins in other Nematode species and showed that the region is highly conserved. LxxLL motifs icated over the amino acid sequences. (B) LIR-2 Protein structure of CRISPR-Cas9 generated mutants. dicates alteration in sequence, triangle indicates insertion, and deletion is marked as a white box. (C) sentative Venus expression in different tissues in mutants were shown. White bars in cr-178 and smo-1 panels indicate position of P5-7.pxx vulval cells. Scale bars: The black bar at the left top applies to all raphs of embryos and the black bar at the left bottom applies to all larval micrographs (50 μm).

Article Snippet: Briefly, CRISPR-Cas9 ribonucleoprotein (RNP) complexes were preassembled by mixing 110 ng/μL tracr RNA (IDT), 100 ng/μL of each guide RNAs (Alt-R crRNA, IDT) and 250 ng/μL Cas9-NLS purified protein (MacroLab, QB3 Berkeley) and incubating at 37 °C for 10 min. 110 ng/μL of each ssODN donor templates (IDT) and 50 ng/μL pRF4: co-injection marker rol-6 (su1006) expression plasmid ( Kramer et al. 1990 ) were then added to the complex mixture.

Techniques: Mutagenesis, CRISPR, Generated, Sequencing, Expressing

Journal: eLife

Article Title: Molecular determinants of phase separation for Drosophila DNA replication licensing factors

doi: 10.7554/eLife.70535

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , 2Cc-T , QB3 Macrolab (UC Berkeley) , RRID: Addgene_37237 , Ligation independent cloning (LIC); E. coli expression vector.

Techniques: Recombinant, Ligation, Cloning, Expressing, Plasmid Preparation

Journal: Nature Ecology & Evolution

Article Title: Evolution of stickleback spines through independent cis -regulatory changes at HOXDB

doi: 10.1038/s41559-022-01855-3

Figure Lengend Snippet: a , Gasterosteus mapping cross. b , QTL scan results for spine number and spine length. x axis: Gasterosteus chromosomes; y axis: LOD score for three- versus four-spine trait (top), length of DS2 (bottom). The QTL peak on chromosome 6 includes the HOXDB cluster (gene diagram at the bottom, scale bar, 1 kb). The peak on chromosome 4 includes the EDA-MSX2A-STC2A cluster described elsewhere , . Dashed lines: genome-wide significance thresholds from permutation testing. c , Integration of GFP reporter using CRISPR–Cas9 upstream of the endogenous HOXD11B locus of low-spine Gasterosteus . Plasmid: grey; eGFP: green; basal hsp70 promoter: blue; chromosomal locus: black. Scale bar, 100 bp. TSS, transcription start site. d , eGFP expression in posterior half of fish at the stage when the dorsal spines are forming (Swarup stage 31). Scale bar, 1 mm. e , Note expression in fin fold between DS2 and DSL, DSL and dorsal fin (DF). Scale bar, 1 mm. f , X-ray of uninjected Gasterosteus (top) and Gasterosteus injected at the single-cell stage with Cas9 and sgRNA targeting the coding region of HOXD11B (bottom). Arrows: two blank pterygiophores are often located between DS2 and DSL but only in uninjected fish (insets: two blank pterygiophores in n = 5 out of 18 control and n = 0 out of 23 injected F0 mutants, two-tailed Fisher’s exact test P = 0.01). Scale bar, 5 mm. g , Length comparisons of dorsal and anal spines. Box and whisker plot: centre line, median; box limits, interquartile range (IQR); whiskers, 1.5× IQR; individual measurements shown as single points (circles: WT; triangles: mutant). y axis: residuals after accounting for standard length of fish (Extended Data Fig. ). DSL and AS were significantly longer in injected than uninjected fish (two-tailed t -test Bonferroni-corrected at α = 0.05, n = 18 control and n = 23 injected, DSL P adj = 3 × 10 −5 , AS P adj = 0.02). DS1 and DS2 lengths were not significantly different.

Article Snippet: Cas9 protein (QB3 MacroLab, University of California at Berkeley), a donor plasmid (pTia1l-hspGFP, deposited at Addgene, containing hsp70, GFP and a single guide RNA (sgRNA) target site), and two sgRNAs were injected.

Techniques: Genome Wide, CRISPR, Plasmid Preparation, Expressing, Injection, Control, Two Tailed Test, Whisker Assay, Mutagenesis

Journal: Nature Ecology & Evolution

Article Title: Evolution of stickleback spines through independent cis -regulatory changes at HOXDB

doi: 10.1038/s41559-022-01855-3

Figure Lengend Snippet: a . X-rays of an uninjected sibling control RABS Gasterosteus (top) and a RABS Gasterosteus that was injected at the single cell stage with Cas9 and an sgRNA targeting the coding region of HOXD11B (bottom). Scale bar is 5 mm. b . Quantification of spine length changes. In the box and whisker plot: center line, median; box limits, interquartile range; whiskers, 1.5x interquartile range; each measurement is represented by a single point (circle for wild type and triangle for mutant). DS1 and DS2 were not significantly different between controls and HOXD11B mutant fish. DSL and AS were significantly longer in the F0 mutants compared to the controls (two-tailed t-test Bonferroni-corrected at α = 0.05, DSL p adj = 4E-13, AS p adj = 2E-07, n = 38 injected and n = 30 control from 3 clutches combined). The y-axis is the residual after accounting for the standard length of fish.

Article Snippet: Cas9 protein (QB3 MacroLab, University of California at Berkeley), a donor plasmid (pTia1l-hspGFP, deposited at Addgene, containing hsp70, GFP and a single guide RNA (sgRNA) target site), and two sgRNAs were injected.

Techniques: Control, Injection, Whisker Assay, Mutagenesis, Two Tailed Test