Endogenous Zebrafish Proneural Cre Drivers Generated by CRISPR/Cas9 Short Homology Directed Targeted Integration

Overview

Journal Sci Rep

Specialty Science

Date 2021 Jan 19

PMID 33462297

Citations 10

Authors

Maira P Almeida

Jordan M Welker

Sahiba Siddiqui

Jon Luiken

Stephen C Ekker

Karl J Clark

Jeffrey J Essner

Maura McGrail

Affiliations

Soon will be listed here.

Abstract

We previously reported efficient precision targeted integration of reporter DNA in zebrafish and human cells using CRISPR/Cas9 and short regions of homology. Here, we apply this strategy to isolate zebrafish Cre recombinase drivers whose spatial and temporal restricted expression mimics endogenous genes. A 2A-Cre recombinase transgene with 48 bp homology arms was targeted into proneural genes ascl1b, olig2 and neurod1. We observed high rates of germline transmission ranging from 10 to 100% (2/20 olig2; 1/5 neurod1; 3/3 ascl1b). The transgenic lines Tg(ascl1b-2A-Cre), Tg(olig2-2A-Cre), and Tg(neurod1-2A-Cre) expressed functional Cre recombinase in the expected proneural cell populations. Somatic targeting of 2A-CreERT2 into neurod1 resulted in tamoxifen responsive recombination in the nervous system. The results demonstrate Cre recombinase expression is driven by the native promoter and regulatory elements of the targeted genes. This approach provides a straightforward, efficient, and cost-effective method to generate cell type specific zebrafish Cre and CreERT2 drivers, overcoming challenges associated with promoter-BAC and transposon mediated transgenics.

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