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.
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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.
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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
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DOI: https://doi.org/10.1007/978-1-0716-3016-7_24
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