Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair

Abstract

Sequence-specific nucleases like TALENs and the CRISPR/Cas9 system have greatly expanded the genome editing possibilities in model organisms such as zebrafish. Both systems have recently been used to create knock-out alleles with great efficiency, and TALENs have also been successfully employed in knock-in of DNA cassettes at defined loci via homologous recombination (HR). Here we report CRISPR/Cas9-mediated knock-in of DNA cassettes into the zebrafish genome at a very high rate by homology-independent double-strand break (DSB) repair pathways. After co-injection of a donor plasmid with a short guide RNA (sgRNA) and Cas9 nuclease mRNA, concurrent cleavage of donor plasmid DNA and the selected chromosomal integration site resulted in efficient targeted integration of donor DNA. We successfully employed this approach to convert eGFP into Gal4 transgenic lines, and the same plasmids and sgRNAs can be applied in any species where eGFP lines were generated as part of enhancer and gene trap screens. In addition, we show the possibility of easily targeting DNA integration at endogenous loci, thus greatly facilitating the creation of reporter and loss-of-function alleles. Due to its simplicity, flexibility, and very high efficiency, our method greatly expands the repertoire for genome editing in zebrafish and can be readily adapted to many other organisms.

A Practical Guide to CRISPR/Cas9 Genome Editing in Lepidoptera

“…Pilot work shown here and elsewhere also demonstrates that targeted insertions are possible as well, thus promising even further developments on the near horizon such as protein tagging, reporter constructs, and tissue-specific expression constructs. Right now the main challenge with knockin strategies is the relatively low efficiency rate, although newer technologies such as NHEJ mediated knockin (Auer et al 2014) promise to dramatically improve this. Perhaps the most exciting thing about CRISPR-associated genome editing approaches, though, is the straightforward portability of the technology between species.…”

“…It has been shown that knocking out factors in the NHEJ pathway can enhance the HDR pathway and increase gene targeting efficiency in Bombyx (Ma et al 2014;Zhu et al 2015). Some Cas9-mediated homology-independent knockin approaches have shown higher efficiency rates in zebrafish (Auer et al 2014) and human cell lines (He et al 2016), suggesting NHEJ repair may provide an alternate strategy to improve incorporation of donor DNA in Lepidoptera.…”

The integration characteristics of the exogenous growth hormone gene in a transgenic common carp (Cyprinus carpio L.) with fast-growth performance

“…Recently, site-specific integration has been achieved in model fish using PhiC31 integrase or genome-editing technology [47][48][49], which would avoid nontarget effects that usually occur in fish with randomly integrated transgenes. So far, however, no ideal integration sites, like ROSA26 in mice and humans, have been found in fish.…”

Learning to Fish with Genetics: A Primer on the Vertebrate ModelDanio rerio

“…The crRNA and Cas9 protein form a complex, with the crRNA targeting a specific DNA sequence and the Cas9 protein catalyzing a double-strand break at the target site (Auer et al 2014). The ease and relative low cost of CRISPR tools have made editing of the zebrafish genome accessible to many laboratories.…”