清脆的
Cas9
基因组编辑
斑马鱼
计算生物学
DNA
引导RNA
点突变
突变体
生物
基因敲除
遗传学
基因
作者
Sergey V. Prykhozhij,Jason N. Berman
出处
期刊:Methods in molecular biology
日期:2023-09-06
卷期号:: 279-303
标识
DOI:10.1007/978-1-0716-3401-1_19
摘要
Introduction or knock-in of precise genomic modifications remains one of the most important applications of CRISPR/Cas9 in all model systems including zebrafish. The most widely used type of donor template containing the desired modification is single-stranded DNA (ssDNA), either in the form of single-stranded oligodeoxynucleotides (ssODN) (<150 nucleotides (nt)) or as long ssDNA (lssDNA) molecules (up to about 2000 nt). Despite the challenges posed by DNA repair after DNA double-strand breaks, knock-in of precise mutations is relatively straightforward in zebrafish. Knock-in efficiency can be enhanced by careful donor template design, using lssDNA as template or tethering the donor template DNA to the Cas9-guide RNA complex. Other point mutation methods such as base editing and prime editing are starting to be applied in zebrafish and many other model systems. However, these methods may not always be sufficiently accessible or may have limited capacity to perform all desired mutation knock-ins which are possible with ssDNA-based knock-in methods. Thus, it is likely that there will be complementarity in the technologies used for generating precise mutants. Here, we review and describe a suite of CRISPR/Cas9 knock-in procedures utilizing ssDNA as the donor template in zebrafish, point out the potential challenges and suggest possible approaches for their solution ultimately leading to successful generation of precise mutant lines.
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