CRISPR/Cpf1双切刻系统的构建与活性检测

Construction and Activity Evaluation of CRISPR-Cpf1 Double-nicking System

Cpf1是2类V型CRISPR-Cas系统的单个RNA引导的核酸内切酶,具有切割DNA的核酸酶活性。该系统由Cpf1核酸酶和CRISPR RNA(crRNA)组成,对于如何提高CRISPR/Cpf1系统的基因编辑效率主要集中在增强crRNA的稳定性、设计对PAM要求更简单的Cpf1变体或者鉴定新型Cpf1核酸内切酶。然而,其是否适用于构建双切刻系统还有待验证。因此,本研究旨在设计3种AsCpf1突变体以及成对的crRNA,进行瞬时转染,利用双荧光素酶报告基因检测评估AsCpf1双切刻系统的编辑效率;同时探究人工改造的crRNA能否提高AsCpf1双切刻系统的编辑效率;以及设计6对具有拓展PAMs序列的crRNA靶向位点,评估HkCpf1双切刻系统在哺乳动物基因组中的编辑效率。结果显示:3种AsCpf1突变体均能不同程度地提高报告基因的相对活性,并具有切割DNA单链的能力。在AsCpf1 crRNA发生突变和延伸后,它们仍然具有引导AsCpf1双切刻系统切割DNA单链的能力。HkCpf1不仅可以用做构建双切刻系统还具有较高的切割活性,并且可以识别5’-YTN和5’-TYYN PAMs序列,与AsCpf1相比,显著拓宽了其靶向范围。

Cpf1 is a single RNA-guided nucleic acid endonuclease of the class II type V CRISPR-Cas system with nuclease activity for cutting DNA. The system consists of Cpf1 nuclease and CRISPR RNA(crRNA). How to improve the gene editing efficiency of CRISPR/Cpf1 system mainly focuses on enhancing the stability of crRNA, designing Cpf1 variants that require simpler PAM, or identifying novel Cpf1 endonucases. However, whether it is suitable for constructing double-nicking system remains to be verified. Therefore, this study aimed to design three AsCpf1 mutants and pairs of crRNA for transient transfection, and evaluate the editing efficiency of the AsCpf1 double-nicking system by using the dual-luciferase reporter gene assay. Meanwhile, whether artificially modified crRNA could improve the editing efficiency of AsCpf1 double-nicking system was explored;Six pairs of crRNA targeting sites with expanded PAMs sequences were designed to evaluate the editing efficiency of HkCpf1 double-nicking system in mammalian genomes. The results showed that all the three AsCpf1 mutants could improve the relative activity of the reporter gene to different degrees and had the ability to cleave DNA single strands.After mutation and extension of the AsCpf1 crRNA, they still have the ability to guide the AsCpf1 nickase to cut the single strand of DNA. HkCpf1 could be used to construct a double-nicking system with high cleavage activity and could recognize target sites with 5’-YTN and 5’-TYYN PAMs, which significantly broadens the target range compared to AsCpf1.