Enhanced Cas12a editing in mammalian cells and zebrafish
Enhanced Cas12a editing in mammalian cells and zebrafish
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DOI：
10.1093/nar/gkz184
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发表时间：
2019-05-07
影响因子：
14.9
通讯作者：
Wolfe, Scot A.
中科院分区：
文献类型：
--
作者：
Liu, Pengpeng;Luk, Kevin;Wolfe, Scot A.
V型CRISPR-Cas12a系统为Cas9提供了一个替代核酸酶的平台，在特定的基因组编辑应用中具有潜在的优势。在这里，我们描述了对Cas12a系统的改进，该系统有助于在哺乳动物细胞和斑马鱼胚胎中进行高效的靶向突变。我们发现，在C端具有两个不同核定位序列(NLS)的Cas12a的工程变体在哺乳动物细胞中提供了更高的编辑效率。此外，我们发现，与标准的成熟crRNA框架相比，包含全长直接重复(Full-DR-crRNA)序列并具有特定的茎环G-C碱基替换的前crRNA显示出更高的编辑效率。最后，我们在斑马鱼胚胎中证明了改良的LbCas12a和FnoCas12a核酸酶与这些修饰的crRNAs相结合，在高浓度下以核糖核蛋白复合体的形式输送时，表现出高突变效率和低毒性。总之，这些结果定义了一组在脊椎动物系统中具有广泛实用价值的增强型Cas12a组件。
Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the improved LbCas12a and FnoCas12a nucleases in combination with these modified crRNAs display high mutagenesis efficiencies and low toxicity when delivered as ribonucleoprotein complexes at high concentration. Together, these results define a set of enhanced Cas12a components with broad utility in vertebrate systems.