Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans.
Robust Genome Editing with Short Single-Stranded and Long, Partially Single-Stranded DNA Donors in Caenorhabditis elegans.
复制标题
DOI：
10.1534/genetics.118.301532
复制
发表时间：
2018-11
期刊：
影响因子：
3.3
通讯作者：
Mello CC
中科院分区：
文献类型：
--
作者：
Dokshin GA;Ghanta KS;Piscopo KM;Mello CC
强大的基因组编辑管道对于现代基因实验室的活力至关重要。先前的研究表明，基于Cas9核糖核蛋白（RNP）的编辑在秀丽隐杆线虫中非常有效，特别是在线虫中。使用核糖核蛋白复合物和合成的单链寡脱氧核苷酸（ssODN）供体的基于CRISPR的基因组编辑可以非常有效。然而，再现性可以变化，并且更长的构建体（例如绿色荧光蛋白标签）的精确的靶向整合在许多系统中仍然具有挑战性。在这里，我们描述了一个精简和优化的编辑协议线虫秀丽隐杆线虫。我们通过亲和标记C. elegans使用ssODN供体。此外，我们描述了一种新的基于PCR的，部分单链的，“杂交”供体设计，产生高效率的编辑与大（内切酶规模）的结构。我们使用这些杂交供体将荧光蛋白标签引入多个位点，实现了接近先前仅用短得多的ssODN供体获得的编辑效率。这里描述的原则和策略可能会转化为其他系统，并应允许研究人员可重复和有效地获得长和短的精确基因组编辑。
A robust genome editing pipeline is critical to the vitality of a modern genetic laboratory. Previous studies have shown that Cas9 ribonucleoprotein (RNP)-based editing can be highly effective in Caenorhabditis elegans, particularly... CRISPR-based genome editing using ribonucleoprotein complexes and synthetic single-stranded oligodeoxynucleotide (ssODN) donors can be highly effective. However, reproducibility can vary, and precise, targeted integration of longer constructs—such as green fluorescent protein tags remains challenging in many systems. Here, we describe a streamlined and optimized editing protocol for the nematode Caenorhabditis elegans. We demonstrate its efficacy, flexibility, and cost-effectiveness by affinity-tagging 14 Argonaute proteins in C. elegans using ssODN donors. In addition, we describe a novel PCR-based, partially single-stranded, “hybrid” donor design that yields high efficiency editing with large (kilobase-scale) constructs. We use these hybrid donors to introduce fluorescent protein tags into multiple loci, achieving editing efficiencies that approach those previously obtained only with much shorter ssODN donors. The principals and strategies described here are likely to translate to other systems, and should allow researchers to reproducibly and efficiently obtain both long and short precision genome edits.
登录
查看更多内容
影响因子：
14.9
作者：
Brinkman EK;Chen T;Amendola M;van Steensel B
通讯作者：
van Steensel B
影响因子：
3.3
作者：
Cho SW;Lee J;Carroll D;Kim JS;Lee J
通讯作者：
Lee J
影响因子：
2.6
作者：
Prior, Harriet;Jawad, Ali K.;Beg, Asim A.
通讯作者：
Beg, Asim A.
影响因子：
30.8
作者：
Richardson, Chris D.;Kazane, Katelynn R.;Corn, Jacob E.
通讯作者：
Corn, Jacob E.
影响因子：
3.3
作者：
Paix A;Folkmann A;Rasoloson D;Seydoux G
通讯作者：
Seydoux G