Efficient single-copy HDR by 5' modified long dsDNA donors.
Efficient single-copy HDR by 5' modified long dsDNA donors.
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DOI：
10.7554/elife.39468
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发表时间：
2018-08-29
期刊：
影响因子：
7.7
通讯作者：
Wittbrodt J
中科院分区：
文献类型：
--
作者：
Gutierrez-Triana JA;Tavhelidse T;Thumberger T;Thomas I;Wittbrodt B;Kellner T;Anlas K;Tsingos E;Wittbrodt J
CRISPR/Cas9通过非同源末端连接有效地诱导靶向突变，但对于基因组编辑，内源DNA片段的精确同源定向修复（HDR）是先决条件。为了支持HDR，许多方法干扰修复机制或操纵Cas9本身。使用Medaka，我们表明长dsDNA供体的5'端的修饰强烈增强HDR，通过保留单体供体构象有利于有效的单拷贝整合，从而促进成功的基因置换或标记。CRISPR/Cas9技术彻底改变了研究人员编辑任何基因组已经测序的生物体DNA的能力。在编辑过程中，RNA的一部分充当向导，以匹配目标DNA的位置。然后，Cas9酶在这个特定位置切割DNA的两条链。新的DNA片段可以被引入细胞，并入DNA“模板”中。细胞使用模板来帮助它修复双链断裂，并在此过程中将新的DNA片段添加到生物体的基因组中。CRISPR/Cas9的一个缺点是，它通常会将新DNA片段的多个拷贝引入基因组中，因为模板可以在粘贴到位之前相互结合。此外，在编辑过程中，新DNA片段的某些部分可能会被遗漏。然而，CRISPR/Cas9的大多数应用-例如，用工作版本替换缺陷基因-需要将所需DNA的一个完整拷贝插入基因组中。为了实现更准确的CRISPR/Cas9基因组编辑，Gutierrez-Triana、Tavhelidse、Thumberger等人将额外的分子连接到DNA模板的末端，以保护DNA在编辑过程中免受错误的影响。修改后的模板被用来耦合干细胞基因的报告，产生一个绿色荧光蛋白到鱼胚胎的基因组。荧光蛋白使得识别偶联成功变得容易。Gutierrez-Triana等人发现，额外的分子可以防止多个模板端对端连接在一起，并确保完整的DNA片段插入基因组中。此外，实验结果表明，只有一个拷贝的模板插入到鱼的DNA中。在未来，新模板将允许在基础研究和治疗应用中以更可控的方式编辑DNA。
CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5’ ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging. CRISPR/Cas9 technology has revolutionized the ability of researchers to edit the DNA of any organism whose genome has already been sequenced. In the editing process, a section of RNA acts as a guide to match up to the location of the target DNA. The enzyme Cas9 then makes a cut in both strands of the DNA at this specific location. New segments of DNA can be introduced to the cell, incorporated into DNA ‘templates’. The cell uses the template to help it to heal the double-strand break, and in doing so adds the new DNA segment into the organism’s genome. A drawback of CRISPR/Cas9 is that it often introduces multiple copies of the new DNA segment into the genome because the templates can bind to each other before being pasted into place. In addition, some parts of the new DNA segment can be missed off during the editing process. However, most applications of CRISPR/Cas9 – for example, to replace a defective gene with a working version – require exactly one whole copy of the desired DNA to be inserted into the genome. In order to achieve more accurate CRISPR/Cas9 genome editing, Gutierrez-Triana, Tavhelidse, Thumberger et al. attached additional molecules to the end of the DNA template to shield the DNA from mistakes during editing. The modified template was used to couple a stem cell gene to a reporter that produces a green fluorescent protein into the genome of fish embryos. The fluorescent proteins made it easy to identify when the coupling was successful. Gutierrez-Triana et al. found that the additional molecules prevented multiple templates from joining together end to end, and ensured the full DNA segment was inserted into the genome. Furthermore, the results of the experiments showed that only one copy of the template was inserted into the DNA of the fish. In the future, the new template will allow DNA to be edited in a more controlled way both in basic research and in therapeutic applications.