Abstract
Clusters of regularly interspaced short palindromic repeats (CRISPR)/Cas systems have a powerful ability to edit DNA and RNA targets. However, the need for a specific recognition site, protospacer adjacent motif (PAM), of the CRISPR/Cas system limits its application in gene editing. Some Argonaute (Ago) proteins have endonuclease functions under the guidance of 5′ phosphorylated or hydroxylated guide DNA (gDNA). The NgAgo protein might perform RNA gene editing at 37 °C, suggesting its application in mammalian cells; however, its mechanisms are unclear. In the present study, the target of NgAgo in RNA was confirmed in vitro and in vivo. Then, an in vitro RNA cleavage system was designed and the cleavage site was verified by sequencing. Furthermore, NgAgo and gDNA were transfected into cells to cleave an intracellular target sequence. We demonstrated targeted degradation of GFP, HCV, and AKR1B10 RNAs in a gDNA-dependent manner by NgAgo both in vitro and in vivo, but no effect on DNA was observed. Sequencing demonstrated that the cleavage sites are located at the 3′ of the target RNA which is recognized by 5′ sequence of the gDNA. These results confirmed that NgAgo–gDNA cleaves RNA not DNA. We observed that the cleavage site is located at the 3′ of the target RNA, which is a new finding that has not been reported in the past.
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Acknowledgements
The authors are very grateful to Mr. Zhang Jian in the Translational Medicine Institute of the First People’s Hospital of Chen Zhou for his kind help with the GFP-positive cell analysis by flow cytometry.
Funding
This work was funded by the Hunan Province Science Fund for Distinguished Young Scholars (2018JJ1021), the Natural Science Foundation of Hunan Province (2017JJ2004), the Hunan Province Technological Innovation Guiding Plan, Clinical Medical Technology Innovation Guidance Project (2017SK51101), the Health Department project of the Hunan Province (B2017182, B20180378, B20180266, and C20180166), the Science and Technique Foundation of Chenzhou (jsyf2017021, jsyf2017023, and zdyf201838), the Key Projects of Chenzhou Science and Technology Foundation (zdyf2020008 and zdyf2020011), and the Project of Xiangnan University of 2017 to ZH.
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ZH, KL, and DXL conceived the study; JYQ, YLX, ZYG, XTL, JJ, TC, and ZH conducted the experiments; ZH, KL, and DXL analyzed the data; JYQ, YLX, and ZH wrote the paper; JYQ and ZH revised the manuscript. All authors read and approved the final manuscript.
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Qu, J., Xie, Y., Guo, Z. et al. Identification of a Novel Cleavage Site and Confirmation of the Effectiveness of NgAgo Gene Editing on RNA Targets. Mol Biotechnol 63, 1183–1191 (2021). https://doi.org/10.1007/s12033-021-00372-1
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DOI: https://doi.org/10.1007/s12033-021-00372-1