Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems

  • Han Wu
  • Qishuai Liu
  • Hui Shi
  • Jingke Xie
  • Quanjun Zhang
  • Zhen Ouyang
  • Nan Li
  • Yi Yang
  • Zhaoming Liu
  • Yu Zhao
  • Chengdan Lai
  • Degong Ruan
  • Jiangyun Peng
  • Weikai Ge
  • Fangbing Chen
  • Nana Fan
  • Qin Jin
  • Yanhui Liang
  • Ting Lan
  • Xiaoyu Yang
  • Xiaoshan Wang
  • Zhiyong Lei
  • Pieter A. Doevendans
  • Joost P. G. Sluijter
  • Kepin Wang
  • Xiaoping Li
  • Liangxue Lai
Original Article

Abstract

CRISPR/Cpf1 features a number of properties that are distinct from CRISPR/Cas9 and provides an excellent alternative to Cas9 for genome editing. To date, genome engineering by CRISPR/Cpf1 has been reported only in human cells and mouse embryos of mammalian systems and its efficiency is ultimately lower than that of Cas9 proteins from Streptococcus pyogenes. The application of CRISPR/Cpf1 for targeted mutagenesis in other animal models has not been successfully verified. In this study, we designed and optimized a guide RNA (gRNA) transcription system by inserting a transfer RNA precursor (pre-tRNA) sequence downstream of the gRNA for Cpf1, protecting gRNA from immediate digestion by 3′-to-5′ exonucleases. Using this new gRNAtRNA system, genome editing, including indels, large fragment deletion and precise point mutation, was induced in mammalian systems, showing significantly higher efficiency than the original Cpf1-gRNA system. With this system, gene-modified rabbits and pigs were generated by embryo injection or somatic cell nuclear transfer (SCNT) with an efficiency comparable to that of the Cas9 gRNA system. These results demonstrated that this refined gRNAtRNA system can boost the targeting capability of CRISPR/Cpf1 toolkits.

Keywords

CRISPR/Cpf1 gRNAtRNA system Genome editing Rabbit Pig 

Notes

Acknowledgements

This work was supported by Grants from the National Natural Science Foundation of China (81702115, 81672317), National Key R&D Program of China (2017YFA0105103, 2017YFA0105101), Bureau of International Cooperation, The Chinese Academy of Sciences (154144KYSB20150033), the Science and Technology Planning Project of Guangdong Province, China (2014B020225003, 2016A030303046, 2015B020229002, 2016A030313169, 2016B030229008), the Youth Innovation Promotion Association, CAS (2017409), Pearl River S&T Nova Program of Guangzhou (201710010112), the Bureau of Science and Technology of Guangzhou Municipality (201704030034), the Science and Technology Planning Project of Guangdong Province, China (2017B030314056).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

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Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Han Wu
    • 1
    • 2
    • 3
  • Qishuai Liu
    • 1
    • 2
    • 3
  • Hui Shi
    • 1
    • 2
    • 3
  • Jingke Xie
    • 1
    • 2
    • 3
  • Quanjun Zhang
    • 1
    • 3
  • Zhen Ouyang
    • 1
    • 3
  • Nan Li
    • 1
    • 2
    • 3
  • Yi Yang
    • 5
  • Zhaoming Liu
    • 1
    • 3
  • Yu Zhao
    • 1
    • 3
  • Chengdan Lai
    • 1
    • 3
  • Degong Ruan
    • 1
    • 2
    • 3
  • Jiangyun Peng
    • 1
    • 2
    • 3
  • Weikai Ge
    • 1
    • 2
    • 3
  • Fangbing Chen
    • 1
    • 2
    • 3
  • Nana Fan
    • 1
    • 3
  • Qin Jin
    • 1
    • 2
    • 3
  • Yanhui Liang
    • 1
    • 2
    • 3
  • Ting Lan
    • 1
    • 2
    • 3
  • Xiaoyu Yang
    • 1
    • 6
  • Xiaoshan Wang
    • 1
    • 2
    • 3
  • Zhiyong Lei
    • 7
    • 8
  • Pieter A. Doevendans
    • 7
    • 8
  • Joost P. G. Sluijter
    • 7
    • 8
  • Kepin Wang
    • 1
    • 3
  • Xiaoping Li
    • 1
    • 3
  • Liangxue Lai
    • 1
    • 3
    • 4
  1. 1.CAS Key Laboratory of Regenerative Biology, Joint School of Life SciencesGuangzhou Institutes of Biomedicine and Health, Guangzhou Medical University, Chinese Academy of SciencesGuangzhouChina
  2. 2.University of Chinese Academy of SciencesBeijingChina
  3. 3.Guangdong Provincial Key Laboratory of Stem Cell and Regenerative MedicineSouth China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of SciencesGuangzhouChina
  4. 4.Jilin Provincial Key Laboratory of Animal Embryo Engineering, Institute of Zoonosis, College of Veterinary MedicineJilin UniversityChangchunChina
  5. 5.Key Laboratory for Major Obstetric Diseases of Guangdong Province, Key Laboratory of Reproduction and Genetics of Guangdong Higher Education InstitutesThe Third Affiliated Hospital of Guangzhou Medical UniversityGuangzhouChina
  6. 6.Institute of Physical Science and Information TechnologyAnhui UniversityHefeiChina
  7. 7.Department of Cardiology, Experimental Cardiology LaboratoryUniversity Medical Center UtrechtUtrechtThe Netherlands
  8. 8.Netherlands Heart InstituteUtrechtThe Netherlands

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