Journal: Gut Microbes

Article Title: Vitamin D influences gut microbiota and acetate production in zebrafish ( Danio rerio ) to promote intestinal immunity against invading pathogens

doi: 10.1080/19490976.2023.2187575

Figure Lengend Snippet: IL-22 mediated VD-induced β-defensin expression in zebrafish intestine. (a) The deletion site by CRISPR/Cas9 on the il22 gene exon (E)1 (exons are in blue boxes) was displayed. (b) The protein level of IL-22 in the intestine of WT and il22 -/- zebrafish was compared ( n = 6/group). The image is representative of 6 replicates. (c) Zebrafish at 3 mpf were i.p . injected with 10 7 CFU E. tarda or PBS, and the survival rate was recorded until 96 hours-post infection ( n = 10/group). (d) The gene expression of zfbd1 , zfbd2 and zfbd3 in zebrafish intestine was measured. (e) After WT and il22 mutant zebrafish at 2 mpf were fed with 0 or 800 IU/kg dietary VD 3 for 4 weeks, the transcript levels of zfbd1 , zfbd2 and zfbd3 in zebrafish intestine were evaluated ( n = 6–8/group). * p < 0.05, *** p < 0.001, ns: non-significance. See also Figures S2.

Article Snippet: The mixture of two sgRNAs was microinjected into one-cell stage embryos together with Cas9 protein (NEB, M0646T, USA) at a final concentration of 1 μg/μL.

Techniques: Expressing, CRISPR, Injection, Infection, Mutagenesis

Journal: bioRxiv

Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 DNA editing tool was used to generate deletions (green box) in the highly conserved DBD from vxs1 (top) and vsx2 (bottom) TFs. Blue boxes represent gene exons, black boxes the location of sgRNAs used to guide Cas9 endonuclease and primers for screening are depicted as opposing arrowheads. b-d and f-h. Histological sections stained with nuclear marker DAPI and phalloidin-Alexa488 for actin filaments from WT (b-d, n≥8) and vsx KO central retinas (f-h, n≥10) at 48hpf (b, f), 72hpf (c, g) and 6dpf (d, h). e, i. Head dorsal view from 6dpf WT (e) and vsx KO (i) larvae with insets showing their pigmentation pattern (white arrowhead). ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar in b-d and f-h: 50μm, scale bar in e and i: 500μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR, Staining, Marker

Journal: bioRxiv

Article Title: Mutation of Vsx genes in zebrafish highlights the robustness of the retinal specification network

doi: 10.1101/2022.01.20.477122

Figure Lengend Snippet: a. CRISPR/Cas9 was used to eliminate (green box) the DBD from vxs1 (top) and vsx2.1 (bottom) TFs in medaka. Blue boxes represent exons, black boxes the location of sgRNAs used and primers for screening are depicted as opposing arrowheads. b-e. Histological sections from WT (b, d, n=4) and vsx KO central retinas (c, e, n=5) at 12dpf. ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer, hpf: hours post-fertilization, dpf: days post-fertilization. Scale bar b-c: 50μm, d-e: 20 μm.

Article Snippet: To target individual vsx genes, a solution containing two sgRNAs (40 ng/μL each) and Cas9 protein (250 ng/μL) (Addgene; 47327) ( ) were injected into one-cell-stage zebrafish and medaka embryos.

Techniques: CRISPR

Journal: FEBS Open Bio

Article Title: Tollip‐deficient zebrafish display no abnormalities in development, organ morphology or gene expression in response to lipopolysaccharide

doi: 10.1002/2211-5463.13423

Figure Lengend Snippet: CRISPR/Cas9‐based genome editing allows for the generation of a Tollip‐deficient zebrafish line. (A) Schemes of the zebrafish tollip transcript variants 1 and 2 (v1 and v2), based on the Ensembl database, showing exons (E), translated sequences (gray), and UTR regions (white). (B) Schematic illustration of the structure of Tollip protein isoforms with the C2 and CUE domains indicated. (C) Partial DNA sequence of the target site within exon 2 of the tollip gene in wild‐type tollip +/+ fish (left) and homozygous tollip −/− knockout fish (right). Deletion of eight nucleotides observed in the mutant line is shadowed in dark gray. There is an additional nucleotide change flanking the deletion (double peak marked R in the chromatogram, corresponding to A or G, with a predicted amino acid change D to G in the truncated protein product), indicating mosaicism of the generated line. (D) Schematic illustration of the predicted structure of Tollip protein isoforms synthesized from the mutated tollip gene. (E) Western blot of the 5 dpf protein lysates from the wild‐type ( tollip +/+ ) line and tollip −/− siblings. Top panel shows Tollip (~ 35 kDa) and a bottom panel shows α‐tubulin (~ 55 kDa) signal. (F) qPCR analysis of the expression of tollip transcripts during early zebrafish development (1–5 dpf). Bars represent the means ± SEM from 3–4 independent experiments (encompassing a pool of 10 larvae/condition). Mann–Whitney U test, * P < 0.05, ****P < 0.0001.

Article Snippet: The Cas9 RNA was synthesized from pCS2‐nCas9n (a gift from Wenbiao Chen, Addgene plasmid, #47929 [ ]) using mMESSAGE mMACHINE T7 Transcription Kit (Life Technologies, #AM1344M).

Techniques: CRISPR, Sequencing, Knock-Out, Mutagenesis, Generated, Synthesized, Western Blot, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Antagonism between regular and atypical Cxcr3 receptors regulates macrophage migration during infection and injury in zebrafish

doi: 10.1101/719526

Figure Lengend Snippet: A 46 bp deletion was induced in the cxcr3.3 gene using CRISPR-Cas9 technology (A) . The deletion is located in the first exon (orange), at the very end of the first transmembrane domain (TM1).The mutation shifts the reading frame and results in a premature stop codon (B) . Nonsense-mediated decay assessment suggests that the cxcr3.3 mutant gene codes a truncated Cxcr3.3 protein ( C ). No evident morphological aberrations were observed in cxcr3.3-/- larvae within the first 5 dpf and the mutant allele segregated following Mendelian proportions (D) . Macrophage development was faster in cxcr3.3-/- embryos at 2 dpf but reverted to WT and cxcr3.2-/- pace after day 3 (E) . Fewer macrophages were found in the head area of cxcr3.2-/- larvae only at day 4 (F) , while there were more macrophages in the tail region in cxcr3.3-/- (G) . The cell numbers corresponding to each day are the average of 35 larvae of each of the 3 groups (genotypes). Data were analyzed using a two-way ANOVA and are shown as mean ± SEM (ns p > 0.05, * p ≤ 0.05, **p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).

Article Snippet: The sgRNA was generated using the MEGA short script ®T7 kit (AM1354, ThermoFisher) and the mRNA for a zebrafish optimized NLS-Cas9-NLS was transcribed using the mMACHINE® SP6 Transcription Kit (AM1340, Thermo Fisher) from a Cas9 plasmid (39312, Addgene) in both cases, the RNeasy Mini Elute Clean up kit (74204, QIAGEN Benelux B.V., Venlo, Netherlands) was used to purify the products.

Techniques: CRISPR, Mutagenesis

Journal: Fishes

Article Title: Editing the Melanocortin-4 Receptor Gene in Channel Catfish Using the CRISPR-Cas9 System

doi: 10.3390/fishes8020116

Figure Lengend Snippet: Figure 5. Embryo survival, hatchability, and fry survival curves of channel catfish, Ictalurus punctatus, embryos from the Kansas random strain microinjected at the one-cell stage with sgRNAs/Cas9 protein targeting the melanocortin-4 receptor (mc4r) gene. Three gRNAs were microinjected individually (MC4RA, MC4RB, and MC4RC) or multiplexed (MC4RMIX). Two control treatments were used. Injected control embryos (iCTRL) were full-sib to the treatment groups and injected with the same solution and volume, but without sgRNA or Cas9 protein. The second control was not injected (nCTRL). (A) Embryo cumulative survival was considered 100% at day 0 then decreased over time as embryos died. (B) The hatch rate was calculated as the fraction of embryos that hatched at a given time point compared to the total live embryos that hatched. (C) Fry survival was calculated for the fry just after hatching and until 20 days post fertilization (dpf).

Article Snippet: Design of the CRISPR-Cas9 System Small guided RNAs (sgRNA) were designed (Table 1) and cloned into a pU6dsgRNA (No. 5) plasmid [42] from Addgene (Cambridge, MA, USA).

Techniques: Control, Injection

Journal: Fishes

Article Title: Editing the Melanocortin-4 Receptor Gene in Channel Catfish Using the CRISPR-Cas9 System

doi: 10.3390/fishes8020116

Figure Lengend Snippet: Figure 6. CRISPR-Cas9-induced mutagenesis in the melanocortin-4 receptor (mc4r) gene of channel catfish, Ictalurus punctatus, Kansas random strain through microinjection of guide RNAs and Cas9 enzyme. The partial sequence of wild-type channel catfish mc4r gene (WT) is the top sequence in each panel. Sequences in green and blue colors are the target sites of the sgRNAs followed by the protospacer-adjacent motif (PAM) sequence, respectively. Red arrows indicate the expected sites of cleavage by Cas9. Dashes indicate the deletion of nucleotides along the mc4r gene. The plus (+) and minus (−) signs indicate insertions and deletions, respectively. (A) MC4RA treatment, (B) MC4RB treatment, (C) MC4RC treatment, and (D) MC4RMIX treatment.

Article Snippet: Design of the CRISPR-Cas9 System Small guided RNAs (sgRNA) were designed (Table 1) and cloned into a pU6dsgRNA (No. 5) plasmid [42] from Addgene (Cambridge, MA, USA).

Techniques: CRISPR, Mutagenesis, Microinjection, Sequencing