June 2022
Volume 63, Issue 7
Open Access
ARVO Annual Meeting Abstract  |   June 2022
An Effective and Versatile Zebrafish Model Using CRISPR Interference to Investigate Gene Functions Regulating Eye Development.
Author Affiliations & Notes
  • Sunit Dutta
    OGVFB, National Eye Institute, Bethesda, Maryland, United States
  • Guangpu Shi
    OGVFB, National Eye Institute, Bethesda, Maryland, United States
    Genetic Engineering, National Eye Institute, Bethesda, Maryland, United States
  • Lijin Dong
    Genetic Engineering, National Eye Institute, Bethesda, Maryland, United States
  • Brian Patrick Brooks
    OGVFB, National Eye Institute, Bethesda, Maryland, United States
  • Footnotes
    Commercial Relationships   Sunit Dutta None; Guangpu Shi None; Lijin Dong None; Brian Brooks None
  • Footnotes
    Support  NONE
Investigative Ophthalmology & Visual Science June 2022, Vol.63, 1357 – F0288. doi:
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      Sunit Dutta, Guangpu Shi, Lijin Dong, Brian Patrick Brooks; An Effective and Versatile Zebrafish Model Using CRISPR Interference to Investigate Gene Functions Regulating Eye Development.. Invest. Ophthalmol. Vis. Sci. 2022;63(7):1357 – F0288.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Antisense Morpholinos ologoneucleotides (MO) has been routinely used in genetic functional studies in zebrafish embryos. However, it’s well known that MO activate p53-associated genes to induce apoptosis. This study was aimed to generate transgenic zebrafish models using CRISPR Interference (CRISPR-i) to interrogate gene functions during eye development.

Methods : Zebrafish-codon-optimized dCas9 was generated from a zebrafish-codon-optimized Cas9 through mutagenesis of D10A and H840A in HNH and RuvC domains respectively. The dCas9 was linked to a synthesized zebrafish- codon-optimized Krab domain and reporter. Using Gateway cloning technology, we generated the following three TolII plasmid systems, Ubi-dCas9-Krab-GFP, Ubi-GFP, and Hsp70-dCas9-Krab-GFP and coinjected these recombinant plasmids and transposes mRNA into zebrafish embryos and screened for founder fish by reporter expression and genotyping. We studied the eye phenotypes in these transgenic embryos expressing dcas9-Krab-GFP by injecting chemically modified-sgRNAs specific for the genes important for eye morphogenesis.

Results : With repeated experiments, we verified that there was no detectable cutting activity of both HNH and RuvC domains of zebrafish-codon-optimized dCas9, and further verified the repressive activity of the synthesized zebrafish-codon-optimized dCas9-Krab system in cultured HEK293 cells using sgRNAs targeting Pax6 and Otx2. rx3 sgRNA injected transgenic embryos expressing dcas9-Krab-GFP developed microphthalmia and/or coloboma at as early as 24 hour post fertilization (hpf) compared to uninjected control transgenic embryos. The microphthalmia and colobama persisted through the observations after 48 hours post fertilization (hpf).

Conclusions : Our experiments indicated that CRISPR-i can be used as a transgenic system in zebrafish and this system is of great value in the investigation of gene functions in ocular morphorgenesis, given the ease of use, versatility for the targets and the short period of time (~24 hrs) needed for induction of ocular phenotypes. This system will allow more rapid ascertainment of phenotypes associated with gene expression screens and paves the way for generating a model system to knock down gene expression in a spatiotemporally regulatable manner.

This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.

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