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Review
, 150, 3-10

Fishing for Understanding: Unlocking the Zebrafish Gene Editor's Toolbox

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Review

Fishing for Understanding: Unlocking the Zebrafish Gene Editor's Toolbox

Brandon W Simone et al. Methods.

Abstract

The rapid growth of the field of gene editing can largely be attributed to the discovery and optimization of designer endonucleases. These include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regular interspersed short palindromic repeat (CRISPR) systems including Cas9, Cas12a, and structure-guided nucleases. Zebrafish (Danio rerio) have proven to be a powerful model system for genome engineering testing and applications due to their external development, high fecundity, and ease of housing. As the zebrafish gene editing toolkit continues to grow, it is becoming increasingly important to understand when and how to utilize which of these technologies for maximum efficacy in a particular project. While CRISPR-Cas9 has brought broad attention to the field of genome engineering in recent years, designer endonucleases have been utilized in genome engineering for more than two decades. This chapter provides a brief overview of designer endonuclease and other gene editing technologies in zebrafish as well as some of their known functional benefits and limitations depending on specific project goals. Finally, selected prospects for additional gene editing tools are presented, promising additional options for directed genomic programming of this versatile animal model system.

Keywords: Base editing; CRISPR; DNA repair; Designer nuclease; Genome editing; Zebrafish.

Figures

Fig 1.
Fig 1.. Established guided designer endonucleases used in zebrafish
(A) Zinc-finger nucleases recognize DNA by the fusion of 3 zinc finger recognition domains on either side of the DNA, specifically binding an 18nt region in this example, and the fused FokI dimerizes and catalyzes cleavage of the DNA. (B) An illustrative 15 repeat TALEN binds on either strand of the DNA separated by a spacer region, and the DSB occurs around the halfway point in the spacer region where the FokI domains dimerize.(C) SpCas9 recognizes the target sequence with the assistance of the sgRNA next to the 3’ PAM sequence and induces a double stranded break 3 base pairs 5’ from the PAM. (D) Cas12a recognizes the target sequence with the assistance of the sgRNA next to the 5’ TTTN PAM sequence and cleaves the DNA at the 18th base on the non-targeted strand and after the 23rd base on the targeted strand to create sticky ends at the DSB. (see text)
Fig 2.
Fig 2.. The structure-guided nuclease
Using a ssDNA guide of at least 20nt, the structure guided nuclease (SGN) dimer cleaves 9–10nt away from the 3’ end of the gDNA on both strands of DNA and creates a large deletion of the intervening genomic locus.
Fig 3.
Fig 3.. The base editor system for non-nuclease editing of the zebrafish genome
The third generation base editor (BE3) catalyzes G:C to an A:T deamination with the fused APOBEC1 nucleobase deaminase. Base excision repair is prevented by the fused uracil glycosylase inhibitor (UGI). The formation of the AT transition is further biased by introducing a nick on the non-edited strand to drive mismatch repair and subsequent resolution to the A:T basepair.

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