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CRISPR/Cas系统从发现和应用以来,热度持续上升.在过去几年中,这种技术已经成为了基因编辑的标准方案.该技术用一条指导RNA(guide RNA)来引导Cas9或Cpf1等核酸酶在特定的位点实现双链打断.但是这种系统也有可能在非特异性的位置进行切割,这种现象成为“脱靶效应”.脱靶剪切现象可能会给基因功能研究或者临床治疗带来很多麻烦.为了深入研究Cpf1的特异性,本研究通过一套双荧光报告系统探索了单核苷酸错配的影响.结果 表明,间隔区内的poly(T)结构会阻止Cpf1的靶向切割.而且rA的错配似乎是CRISPR/Cpf1容忍度最低的一类,这与CRISPR/Cas9的情况相同.这种相似性可能来源于两种酶在进化上的同源性.上述结果表明,在使用CRISPR/Cp1或者CRISPR/Cas9的时候应当更多注意其脱靶效应,因为这种效应是这套系统的一个内在特性.“,”CRISPR/Cas has been coming to prosperity since its discovery and application.It becomes a standard solution for gene editing in the past few years.A guide RNA is used to lead the endonuclease,such as Cas9 and Cpf1,to specific sites and break the double strand.However,there is also possibility that the system will cut a non-specific position,which is called “off-target effect”.The off-target cleavage may cause trouble to gene function research or clinic treatment.In order to reveal the target specificity of Cpf1,this study explored the single-nucleotide mismatches by a dual-luciferase system.Our results showed that the poly(T) structure was prohibitive in spacer for Cpf1 targeting.Moreover,rA mismatches seemed to be of the least tolerance for CRISPR/Cpf1,which was same as CRISPR/Cas9.The phenomenon might be attributed to the homology of the two enzymes.In summary,our research suggest that more attention should be paid to off-target effects when using CRISPR/Cpf1 or CRISPR/Cas9,as this is an intrinsic characteristic of the system.