Precise Editing of the Zebrafish Genome Made Simple and Efficient

Abstract

SUMMARY We present simple and efficient methods for creating heritable modifications of the zebrafish genome. Precisely modified alleles are generated by homologous recombination between the host genome and dsDNA donor molecules, stimulated by the induction of chromosomally targeted DSBs. Several kilobase-long tracts of genome sequence can be replaced. Tagging donor sequences with reporter genes that can be subsequently excised improves recovery of edited alleles by an order of magnitude and facilitates recovery of recessive and phenotypically silent conditional mutations. We generate and demonstrate functionality of: i) alleles with a single codon change, ii) an allele encoding an epitope-tagged version of an endogenous protein, iii) alleles expressing reporter proteins, and iv) a conditional allele in which an exon is flanked by recombinogenic loxP sites. Our methods make recovery of a broad range of genome editing events very practicable, significantly advancing applicability of the zebrafish for studying normal biological processes and modeling diseases.

Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9

“…utilized both TALEN and CRISPR/Cas9 to generate DSB and showed high-efficiency and precise knock-in in zebrafish by using a donor sequence that contained the cargo and was flanked by a 1kb homologous region on either side of the cargo. They used this strategy to tag a gene with V5 tag, insert fluorescent protein gene and insert loxP cassette in either side of an exon of interest in order to produce induced mutants [106].…”

Approaching Perfection: New Developments in Zebrafish Genome Engineering

“…In the tradition of the zebrafish community in Utah, the authors have made available a series of targeting vector backbones with I-Sce1 sites that will be useful for the community as we move forward with HR. Hoshijima et al (2016) put HR to a range of valuable uses, demonstrating the most advanced genome editing in zebrafish to date. They make specific single amino acid changes, fuse endogenous proteins to epitope tags or GFP, make targeted knockins that replace essential exons with GFP, and make Cre-dependent conditional (floxed) alleles.…”

“…In this issue of Developmental Cell, Hoshijima et al (2016) take genome modification in the zebrafish to the next level, demonstrating the efficient use of homologous recombination to make genetic tools for a range of applications.…”

“…To name a few: adding epitopes or fluorescent proteins to endogenous proteins for capture or in vivo localization; generating reporters of endogenous gene expression; modeling human diseases by making mutations analogous to those found in patients; or making landing sites in genes of interest for conditional knockout by site-specific recombinases. The paper by Hoshijima et al (2016), in the current issue of Developmental Cell takes us a major step toward this goal by generating examples of each of these modifications using programmable nucleases to initiate the insertion of specific sequences by homologous recombination (HR).…”

CRISPR/Cas9-Mediated Knockin and Knockout in Zebrafish

“…While this strategy allows keeping the integrity of the targeted coding sequence, the enriched presence of repeat sequences within the introns makes it difficult to achieve a specific targeting. Finally, the latest advance in knockin approaches is the development of traceable genome editing events that allow the easy recovery of edited alleles (Hoshijima et al 2016) (Fig. 1).…”