Efficient homologous recombination-mediated genome engineering in zebrafish using TALE nucleases

Abstract

Custom-designed nucleases afford a powerful reverse genetic tool for direct gene disruption and genome modification in vivo. Among various applications of the nucleases, homologous recombination (HR)-mediated genome editing is particularly useful for inserting heterologous DNA fragments, such as GFP, into a specific genomic locus in a sequence-specific fashion. However, precise HR-mediated genome editing is still technically challenging in zebrafish. Here, we establish a GFP reporter system for measuring the frequency of HR events in live zebrafish embryos. By co-injecting a TALE nuclease and GFP reporter targeting constructs with homology arms of different size, we defined the length of homology arms that increases the recombination efficiency. In addition, we found that the configuration of the targeting construct can be a crucial parameter in determining the efficiency of HR-mediated genome engineering. Implementing these modifications improved the efficiency of zebrafish knock-in generation, with over 10% of the injected F0 animals transmitting gene-targeting events through their germline. We generated two HR-mediated insertion alleles of sox2 and gfap loci that express either superfolder GFP (sfGFP) or tandem dimeric Tomato (tdTomato) in a spatiotemporal pattern that mirrors the endogenous loci. This efficient strategy provides new opportunities not only to monitor expression of endogenous genes and proteins and follow specific cell types in vivo, but it also paves the way for other sophisticated genetic manipulations of the zebrafish genome.

A Defect in DNA Ligase4 Enhances the Frequency of TALEN-Mediated Targeted Mutagenesis in Rice

“…It is widely accepted that DSBs mediated by SSNs at a target gene stimulate GT in several organisms, such as mammals (Pauwels et al, 2014), fish (Shin et al, 2014), insects (Lin et al, 2014), and plants (Puchta and Fauser, 2013;Voytas and Gao, 2014). Here, the induction of DSBs mediated via SSNs in a lig4 mutant background provided evidence that the SDSA-like pathway was enhanced by the disruption of cNHEJ in rice.…”

“…DSB repair via HR using exogenous donor DNA, resulting in the replacement or insertion of new nucleotide sequences, is referred to as gene targeting (GT). Consequently, SSN-mediated DSB induction at the target locus could lead to an increased frequency of GT in several organisms (Puchta and Fauser, 2013;Lin et al, 2014;Pauwels et al, 2014;Shin et al, 2014;Voytas and Gao, 2014).…”

The zebrafish tailbud contains two independent populations of midline progenitor cells that maintain long-term germ layer plasticity and differentiate based on local signaling cues

“…2J, arrowhead). Additionally, an endogenously tagged sox2-p2a-sfGFP reporter line (Shin et al, 2014) exhibits fluorescence in posterior notochord cells, which do not express sox2 transcript or protein, indicating that at least some notochord cells were previously sox2 positive (Fig. 2K,K′, arrowheads).…”

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…In 2013, Zu et al reported the first HR gene-targeting event using TALENs and a double stranded vector containing an eGFP cassette flanked by long homology arms and a germ line transmission rate of 1.5%. More recently many other laboratories have developed various methods to generate knockin alleles by HR followed by CRISPR/Cas9-induced DSB, using as donor single stranded DNA, circular or linear plasmids with short (~40 bp) or long (800-1000 bp) homology arms (Hruscha et al 2013;Hwang et al 2013a;Irion et al 2014;Shin et al 2014;He et al 2015;Hisano et al 2015). Although these methods were proven possible, their efficiency remains variable.…”