Transgenic Expression and Genome Editing by Electroporation of Zebrafish Embryos

Venous thrombosis occurs in patients with quantitative and qualitative fibrinogen disorders. Injury-induced thrombosis in zebrafish larvae has been used to model human coagulopathies. We aimed to determine whether zebrafish models of afibrinogenemia and dysfibrinogenemia have different thrombotic phenotypes. Laser injuries were used to induce venous thrombosis and the time-to-occlusion (TTO) and the binding and aggregation of fluorescent Tg(itga2b:EGFP) thrombocytes measured. The fga−/− larvae failed to support occlusive venous thrombosis and showed reduced thrombocyte binding and aggregation at injury sites. The fga+/− larvae were largely unaffected. When genome editing zebrafish to produce fibrinogen Aα R28C, equivalent to the human Aα R35C dysfibrinogenemia mutation, we detected in-frame skipping of exon 2 in the fga mRNA, thereby encoding AαΔ19–56. This mutation is similar to Fibrinogen Montpellier II which causes hypodysfibrinogenemia. Aα+/Δ19–56 fish had prolonged TTO and reduced thrombocyte activity, a dominant effect of the mutation. Finally, we used transgenic expression of fga R28C cDNA in fga knock-down or fga−/− mutants to model thrombosis in dysfibrinogenemia. Aα R28C expression had similar effects on TTO and thrombocyte activity as Aα+/Δ19–56. We conclude that thrombosis assays in larval zebrafish can distinguish between quantitative and qualitative fibrinogen disorder models and may assist in anticipating a thrombotic phenotype of novel fibrinogen mutations.

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Genome editing with RNA-guided Cas9 nuclease in Zebrafish embryos

Cell Research ◽

10.1038/cr.2013.45 ◽

2013 ◽

Vol 23 (4) ◽

pp. 465-472 ◽

Cited By ~ 493

Author(s):

Nannan Chang ◽

Changhong Sun ◽

Lu Gao ◽

Dan Zhu ◽

Xiufei Xu ◽

...

Keyword(s):

Genome Editing ◽

Zebrafish Embryos ◽

Cas9 Nuclease

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Orthogonal CRISPR-Cas genome editing and efficient inhibition with anti-CRISPRs in zebrafish embryos

10.1101/2020.11.07.372151 ◽

2020 ◽

Author(s):

Paige R. Takasugi ◽

Evan P. Drage ◽

Sahar N. Kanishka ◽

Marissa A. Higbee ◽

James A. Gagnon

Keyword(s):

Genome Editing ◽

Streptococcus Pyogenes ◽

High Efficiency ◽

Bacterial Species ◽

Zebrafish Embryos ◽

Type Ii ◽

Guide Rnas ◽

Highly Effective ◽

Orthogonal Systems ◽

Spatiotemporal Control

AbstractThe CRISPR-Cas universe continues to expand. The type II CRISPR-Cas system from Streptococcus pyogenes (SpCas9) is most widely used for genome editing due to its high efficiency in cells and organisms. However, concentrating on a single CRISPR-Cas system limits options for multiplexed editing. We hypothesized that CRISPR-Cas systems originating from different bacterial species could operate simultaneously and independently due to their distinct single-guide RNAs (sgRNAs) or CRISPR-RNAs (crRNAs), and protospacer adjacent motifs (PAMs). Additionally, we hypothesized that CRISPR-Cas activity in zebrafish could be regulated through the expression of inhibitory anti-CRISPR (Acr) proteins. Here, we use a simple mutagenesis approach to demonstrate that CRISPR-Cas systems from Streptococcus pyogenes (SpCas9), Streptococcus aureus (SaCas9), and Lachnospiraceae bacterium (LbCas12a, previously known as LbCpf1) are highly effective, orthogonal systems capable of operating simultaneously in zebrafish. We also demonstrate that type II Acrs are effective inhibitors of SpCas9 in zebrafish. These results indicate that at least three orthogonal CRISPR-Cas systems and two anti-CRISPR proteins are functional in zebrafish embryos. These CRISPR-Cas systems and Acr proteins will enable combinatorial and intersectional strategies for spatiotemporal control of genome editing in zebrafish.

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Outcomes Comparison of SpCas9- and LbCas12a-mediated DNA Editing in Zebrafish Embryos

10.20944/preprints202005.0253.v1 ◽

2020 ◽

Author(s):

Darya Meshalkina ◽

Aleksei Glushchenko ◽

Elana Kysil ◽

Igor Mizgirev ◽

Andrej Frolov

Keyword(s):

Genome Editing ◽

Mode Of Action ◽

Target Gene ◽

Gene Knockout ◽

The Other ◽

Zebrafish Embryos ◽

Guide Rnas ◽

Research Technology ◽

Ribonucleoprotein Complexes ◽

Dna Editing

CRISPR/Cas genome editing is a widely used research technology. Its simplest variant is gene knockout resulting from reparation errors after introduction of dsDNA breaks by Cas nuclease. We compared the outcomes of the break repair by two commonly used nucleases (SpCas9 and LbCas12a) in zebrafish embryos to reveal if application of one nuclease is advantageous in comparison to the other. To address this question, we injected ribonucleoprotein complexes of nucleases and corresponding guide RNAs in zebrafish zygotes and three days later sequenced the target gene regions. We found that LbCas12a breaks resulted in longer deletions and more rare inserts, in comparison to those generated by SpCas9, while the editing efficiencies of both nucleases were the same. On the other hand, overlapping protospacers were shown to lead to similarities in repair outcome, although they were cut by two different nucleases. Thus, our results indicate that the repair outcome depends both on the nuclease mode of action and on protospacer sequence.

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Centrobin controls primary ciliogenesis in vertebrates

The Journal of Cell Biology ◽

10.1083/jcb.201706095 ◽

2018 ◽

Vol 217 (4) ◽

pp. 1205-1215 ◽

Cited By ~ 12

Author(s):

Yetunde Adesanya Ogungbenro ◽

Teresa Casar Tena ◽

David Gaboriau ◽

Pierce Lalor ◽

Peter Dockery ◽

...

Keyword(s):

Monoclonal Antibody ◽

Genome Editing ◽

Morphological Features ◽

Serum Starvation ◽

Zebrafish Embryos ◽

Terminal Portion ◽

Centrosomal Protein ◽

Centriole Duplication ◽

Microtubule Stability ◽

Mother Centriole

The BRCA2 interactor, centrobin, is a centrosomal protein that has been implicated in centriole duplication and microtubule stability. We used genome editing to ablate CNTROB in hTERT-RPE1 cells and observed an increased frequency of monocentriolar and acentriolar cells. Using a novel monoclonal antibody, we found that centrobin primarily localizes to daughter centrioles but also associates with mother centrioles upon serum starvation. Strikingly, centrobin loss abrogated primary ciliation upon serum starvation. Ultrastructural analysis of centrobin nulls revealed defective axonemal extension after mother centriole docking. Ciliogenesis required a C-terminal portion of centrobin that interacts with CP110 and tubulin. We also depleted centrobin in zebrafish embryos to explore its roles in an entire organism. Centrobin-depleted embryos showed microcephaly, with curved and shorter bodies, along with marked defects in laterality control, morphological features that indicate ciliary dysfunction. Our data identify new roles for centrobin as a positive regulator of vertebrate ciliogenesis.

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