Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
Efficient Generation of Endogenous Fluorescent Reporters by Nested CRISPR in Caenorhabditis elegans
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DOI：
10.1534/genetics.119.301965
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发表时间：
2019-04-01
期刊：
影响因子：
3.3
通讯作者：
Ceron, Julian
中科院分区：
文献类型：
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作者：
Vicencio, Jeremy;Martinez-Fernandez, Carmen;Ceron, Julian
在模式生物中，基于CRISPR的基因组编辑方法正在以惊人的速度发展。尽管缺失或错义突变体的产生非常简单，但内源性荧光报告基因的产生更具挑战性。我们已经开发了Nested CRISPR，这是一种无克隆核糖核蛋白驱动的方法，可在秀丽隐杆线虫中稳健地产生具有EGFP、mCherry或wrmScarlet的内源性荧光报告基因。该方法基于将荧光蛋白（FP）序列分为三个片段。在第一步中，单链DNA（ssDNA）供体（
CRISPR-based genome-editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is more challenging. We have developed Nested CRISPR, a cloning-free ribonucleoprotein-driven method that robustly produces endogenous fluorescent reporters with EGFP, mCherry or wrmScarlet in Caenorhabditis elegans. This method is based on the division of the fluorescent protein (FP) sequence in three fragments. In the first step, single-stranded DNA (ssDNA) donors (