清脆的
反式激活crRNA
基因组编辑
Cas9
生物
斑马鱼
遗传学
计算生物学
基因组
基因靶向
同源定向修复
基因组工程
DNA
基因
移码突变
非同源性末端接合
DNA修复
突变
核苷酸切除修复
作者
Yusuke Kamachi,Atsuo Kawahara
出处
期刊:Methods in molecular biology
日期:2023-01-01
卷期号:: 313-324
被引量:2
标识
DOI:10.1007/978-1-0716-3016-7_24
摘要
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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