Abstract
CRISPR-Cas9 genome editing technology has been successfully applied to generate various genetic modifications in zebrafish. The CRISPR-Cas9 system, which originally consisted of three components, CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9, efficiently induces DNA double-strand breaks (DSBs) at targeted genomic loci, often resulting in frameshift-mediated target gene disruption (knockout). However, it remains difficult to perform the targeted integration of exogenous DNA fragments (knock-in) with CRISPR-Cas9. DSBs can be restored through DNA repair mechanisms, such as nonhomologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). One of our two research groups established a method for the precise MMEJ-mediated targeted integrations of exogenous genes containing homologous microhomology sequences flanking a targeted genomic locus in zebrafish. The other group recently developed a method for knocking in ~200 nt sequences encoding composite tags using long single-stranded DNA (ssDNA) donors. This chapter summarizes the CRISPR-Cas9-mediated genome modification strategy in zebrafish.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hisano Y, Ota S, Kawahara A (2014) Genome editing using artificial site-specific nucleases in zebrafish. Develop Growth Differ 56:26–33
Haffter P, Granato M, Brand M, Mullins MC, Hammerschmidt M, Kane DA et al (1996) The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123:1–36
Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL et al (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123:37–46
Thisse C, Zon LI (2002) Organogenesis-heart and blood formation from the zebrafish point of view. Science 295:457–462
Kawahara A, Nishi T, Hisano Y, Fukui H, Yamaguchi A, Mochizuki N (2009) The sphingolipid transporter spns2 functions in migration of zebrafish myocardial precursors. Science 323:524–527
McVey M, Lee SE (2008) MMEJ repair of double-strand breaks (director’s cut): deleted sequences and alternative endings. Trends Genet 24:529–538
Hoshijima K, Jurynec MJ, Klatt Shaw D, Jacobi AM, Behlke MA, Grunwals DJ (2019) Highly efficient CRISPR-Cas9-based methods for generating deletion mutations and F0 embryos that lack gene function in zebrafish. Dev Cell 51:645–657
Kroll F, Powell GT, Ghosh M, Gestri G, Antinucci P, Hearn TJ et al (2021) A simple and effective F0 knockout method for rapid screening of behaviour and other complex phenotypes. elife 10:e59683
Quick RE, Buck LD, Parab S, Tolbert ZR, Matsuoka RL (2021) Highly efficient synthetic CRISPR RNA/Cas9-based mutagenesis for rapid cardiovascular phenotypic screening in F0 zebrafish. Front Cell Dev Biol 9:735598
Suzuki T, Hirai Y, Uehara T, Ohga R, Kosaki K, Kawahara A (2021) Involvement of the zebrafish trrap gene in craniofacial development. Sci Rep 11:24166
Auer TO, Duroure K, De Cian A, Concordet JP, Del Bene F (2014) Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair. Genome Res 24:142–153
Kimura Y, Hisano Y, Kawahara A, Higashijima S (2014) Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering. Sci Rep 4:6545
Ota S, Taimatsu K, Yanagi K, Namiki T, Higashijima S, Kawahara A (2016) Functional visualization and disruption of targeted genes using CRISPR/Cas9-mediated eGFP reporter integration in zebrafish. Sci Rep 6:1038
Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H et al (2015) Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish. Sci Rep 5:8841
Ranawakage DC, Okada K, Sugio K, Kawaguchi Y, Kuninobu-Bonkohara Y, Takada T et al (2021) Efficient CRISPR-Cas9-mediated knock-in of composite tags in zebrafish using long ssDNA as a donor. Front Cell Dev Biol 8:598634
Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD et al (2013) Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 31:227–229
Ansai S, Kinoshita M (2014) Targeted mutagenesis using CRISPR/Cas system in medaka. Biol Open 3:362–371
Acknowledgments
The works of Kawahara’s group were supported by the Japan Society for the Promotion of Science (JSPS) and the Japan Agency for Medical Research and Development (AMED). The works of Kamachi’s group were supported by JSPS.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Kamachi, Y., Kawahara, A. (2023). CRISPR-Cas9-Mediated Genome Modifications in Zebrafish. In: Hatada, I. (eds) Genome Editing in Animals. Methods in Molecular Biology, vol 2637. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3016-7_24
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3016-7_24
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3015-0
Online ISBN: 978-1-0716-3016-7
eBook Packages: Springer Protocols