SplitAx: A novel method to assess the function of engineered nucleases.
SplitAx: A novel method to assess the function of engineered nucleases.
复制标题
DOI：
10.1371/journal.pone.0171698
复制
发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Forrester LM
中科院分区：
文献类型：
--
作者：
Axton RA;Haideri SS;Lopez-Yrigoyen M;Taylor HA;Forrester LM
工程化核酸酶已用于产生敲除或报告细胞系和一系列人类疾病的动物模型。这些新技术也为治疗性基因组编辑带来了巨大的希望。目前的方法来评估这些核酸酶的活性是耗时的，需要大量的优化，并受到低信号和高背景的读数。我们已经开发了一种简单易行的方法（SplitAx），该方法在很大程度上解决了这些问题，并提供了核酸酶活性的读数。该测定涉及用核酸酶结合位点序列分裂GFP的N-末端（氨基酸1-158）编码区和C-末端区的框外。暴露于测试核酸酶后，通过易出错的非同源末端连接（NHEJ）进行切割和修复，恢复阅读框，从而产生全长荧光GFP蛋白。荧光也可以通过N-末端和C-末端编码序列之间的反式互补来恢复。我们证明了SplitAx测定法的成功使用，以评估锌指核酸酶，CRISPR hCAS 9和TALENS的功能。我们还测试了CRISPR/hCas 9/D10 A系统中多种gRNA的活性。然后将在SplitAx测定中显示功能活性的锌指核酸酶和向导RNA成功用于靶向内源性AAVS 1、S 0X 6和Cfms基因座。这种简单的方法可以应用于其他不相关的蛋白质，如ZsGreen 1，并提供了一个测试系统，不需要复杂的优化。
Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1–158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization.