Chapter title |
CRISPR-Cas9-Mediated Genome Modifications in Zebrafish.
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Chapter number | 24 |
Book title |
Genome Editing in Animals
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Published in |
Methods in molecular biology, January 2023
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DOI | 10.1007/978-1-0716-3016-7_24 |
Pubmed ID | |
Book ISBNs |
978-1-07-163015-0, 978-1-07-163016-7
|
Authors |
Kamachi, Yusuke, Kawahara, Atsuo |
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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