Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.
Comparative analysis of lipid-mediated CRISPR-Cas9 genome editing techniques.
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DOI：
10.1002/cbin.10952
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发表时间：
2018-07
影响因子：
3.9
通讯作者：
Alder JK
中科院分区：
文献类型：
--
作者：
Ringer KP;Roth MG;Garey MS;Piorczynski TB;Suli A;Hansen JM;Alder JK
CRISPR-Cas技术彻底改变了基因组工程。虽然Cas9不是第一个被鉴定的可编程核酸内切酶，但其使用简单性在短时间内推动了广泛采用。虽然CRISPR-Cas基因组编辑在临床应用方面具有巨大的潜力，但它在实验室环境中用于基因型-表型研究和全基因组筛选，导致了对许多分子途径理解的突破。已经描述了许多用于将CRISPR-Cas组分引入细胞的方案，在这里，我们试图使用容易获得且廉价的工具简化和优化基因组编辑的方案。我们比较了质粒、核糖核蛋白（RNP）和RNA转染，以确定哪种方法是在实验室环境中编辑细胞的最佳方法。我们将我们的比较限于脂质体介导的引入，因为试剂是广泛可用的。为了便于优化，我们开发了一种新的报告基因检测方法来测量基因破坏和各种外源DNA标签的引入。每种方法都有效地破坏了内源基因，并能够刺激外源DNA在特定位点的引入，尽管效率不同。RNP转染产生最高水平的基因破坏，并且是总体上最快速和有效的方法。最后，我们表明，30个碱基对的非常短的同源臂可以介导位点特异性编辑。这里描述的方法应该扩大RNP介导的脂质转染用于实验室基因组编辑实验的可及性。
CRISPR-Cas technology has revolutionized genome engineering. While Cas9 was not the first programmable endonuclease identified, its simplicity of use has driven widespread adoption in a short period of time. While CRISPR-Cas genome editing holds enormous potential for clinical applications, its use in laboratory settings for genotype-phenotype studies and genome-wide screens has led to breakthroughs in the understanding of many molecular pathways. Numerous protocols have been described for introducing CRISPR-Cas components into cells, and here we sought to simplify and optimize a protocol for genome editing using readily available and inexpensive tools. We compared plasmid, ribonucleoprotein (RNP), and RNA transfection to determine which was method was most optimal for editing cells in a laboratory setting. We limited our comparison to lipofection-mediated introduction because the reagents are widely available. To facilitate optimization, we developed a novel reporter assay to measure gene disruption and the introduction of a variety of exogenous DNA tags. Each method efficiently disrupted endogenous genes and was able to stimulate the introduction of foreign DNA at specific sites, albeit to varying efficiencies. RNP transfection produced the highest level of gene disruption and was the most rapid and efficient method overall. Finally, we show that very short homology arms of 30 base pairs can mediate site-specific editing. The methods described here should broaden the accessibility of RNP-mediated lipofection for laboratory genome-editing experiments.
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影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE
影响因子：
4.1
作者：
Liang, Xiquan;Potter, Jason;Chesnut, Jonathan D.
通讯作者：
Chesnut, Jonathan D.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
56.9
作者：
Boch, Jens;Scholze, Heidi;Bonas, Ulla
通讯作者：
Bonas, Ulla
通过反式编码的小 RNA 和宿主因子 RNase III 进行 CRISPR RNA 成熟。
影响因子：
64.8
作者：
Deltcheva, Elitza;Chylinski, Krzysztof;Sharma, Cynthia M.;Gonzales, Karine;Chao, Yanjie;Pirzada, Zaid A.;Eckert, Maria R.;Vogel, Joerg;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle