SplitAx: A novel method to assess the function of engineered nucleases

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.工程化核酸酶（Engineered nucleases）已被用于构建人类疾病相关的基因敲除细胞系、报告基因细胞系及多种动物模型。这类新兴技术在治疗性基因组编辑领域同样具有广阔的应用前景。当前用于评估这类核酸酶活性的方法存在耗时长、需大量优化工作的缺陷，且受限于信号偏弱、背景噪声较高的检测信号。本研究开发了一种简便易操作的SplitAx检测方法，该方法可有效解决上述问题，并可获取核酸酶活性的检测信号。该检测方法将绿色荧光蛋白（GFP）的N端（氨基酸1-158）编码区，与带有核酸酶结合位点序列且处于移码状态的C端编码区进行拆分。当该检测系统接触待测试的核酸酶后，核酸酶的切割作用与易出错的非同源末端连接（non-homologous end joining, NHEJ）修复过程可恢复正确的阅读框，最终生成具有荧光活性的全长GFP蛋白。此外，N端与C端编码序列也可通过反式互补作用恢复荧光信号。本研究验证了SplitAx检测方法可成功用于评估锌指核酸酶（zinc finger nucleases）、CRISPR hCas9以及转录激活因子样效应物核酸酶（TALENS）的功能活性。本研究同时在CRISPR/hCas9/D10A系统中测试了多条向导RNA（gRNA）的活性。经SplitAx检测验证具有功能活性的锌指核酸酶与向导RNA，可成功靶向内源AAVS1、SOX6及Cfms基因座。该简便检测方法还可推广至ZsGreen1等其他非相关蛋白的检测，且无需复杂的优化步骤即可完成检测。