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

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.

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References

  1. 1.

    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10(10):957–963

  2. 2.

    Cho SW, Kim S, Kim JM, Kim J-S (2013) Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 31(3):230–232

  3. 3.

    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821

  4. 4.

    Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales APW, Li Z, Peterson RT, Yeh J-RJ et al (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523(7561):481

  5. 5.

    Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z et al (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63

  6. 6.

    Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A et al (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163(3):759–771

  7. 7.

    Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Reports 6:38169

  8. 8.

    Xu R, Qin R, Li H, Li D, Li L, Wei P, Yang J (2017) Generation of targeted mutant rice using a CRISPR-Cpf1 system. Plant Biotechnol J 15(6):713–717

  9. 9.

    Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q et al (2017) A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nature plants 3:17018

  10. 10.

    Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nature Commun 8:7

  11. 11.

    Ungerer J, Pakrasi HB (2016) Cpf1 is a versatile tool for CRISPR genome editing across diverse species of cyanobacteria. Sci Reports 6:39681

  12. 12.

    Port F, Bullock SL (2016) Augmenting CRISPR applications in drosophila with tRNA-flanked sgRNAs. Nat Methods 13(10):852–854

  13. 13.

    Hur JK, Kim K, Been KW, Baek G, Ye S, Hur JW, Ryu SM, Lee YS, Kim JS (2016) Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat Biotechnol 34(8):807–808

  14. 14.

    Kim Y, Cheong SA, Lee JG, Lee SW, Lee MS, Baek IJ, Sung YH (2016) Generation of knockout mice by Cpf1-mediated gene targeting. Nat Biotechnol 34(8):808–810

  15. 15.

    Watkins-Chow DE, Varshney GK, Garrett LJ, Chen Z, Jimenez EA, Rivas C, Bishop KS, Sood R, Harper UL, Pavan WJ et al (2017) Highly efficient Cpf1-mediated gene targeting in mice following high concentration pronuclear injection. G3 7(2):719–722

  16. 16.

    Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS et al (2017) Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol 35(1):31–34

  17. 17.

    Zhang Y, Long C, Li H, McAnally JR, Baskin KK, Shelton JM, Bassel-Duby R, Olson EN (2017) CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice. Sci Adv 3(4):e1602814–e1602814

  18. 18.

    Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK (2016) Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol 34(8):869–874

  19. 19.

    Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS (2016) Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34(8):863–868

  20. 20.

    Yang M, Wei H, Wang Y, Deng J, Tang Y, Zhou L, Guo G, Tong A (2017) Targeted disruption of V600E-mutant BRAF gene by CRISPR-Cpf1. Mol Ther Nucleic Acids 8:450–458

  21. 21.

    Tu M, Lin L, Cheng Y, He X, Sun H, Xie H, Fu J, Liu C, Li J, Chen D et al (2017) A ‘new lease of life’: FnCpf1 possesses DNA cleavage activity for genome editing in human cells. Nucleic Acids Res 45(19):11295–11304

  22. 22.

    Lei C, Li SY, Liu JK, Zheng X, Zhao GP, Wang J (2017) The CCTL (Cpf1-assisted Cutting and Taq DNA ligase-assisted Ligation) method for efficient editing of large DNA constructs in vitro. Nucleic Acids Res 45(9):e74

  23. 23.

    Orlando SJ, Santiago Y, DeKelver RC, Freyvert Y, Boydston EA, Moehle EA, Choi VM, Gopalan SM, Lou JF, Li J (2010) Zinc-finger nuclease-driven targeted integration into mammalian genomes using donors with limited chromosomal homology. Nucleic Acids Res 38(15):e152–e152

  24. 24.

    Song J, Zhong J, Guo XG, Chen YQ, Zou QJ, Huang J, Li XP, Zhang QJ, Jiang ZW, Tang CC et al (2013) Generation of RAG 1-and 2-deficient rabbits by embryo microinjection of TALENs. Cell Res 23(8):1059–1062

  25. 25.

    Yang Y, Wang K, Wu H, Jin Q, Ruan D, Ouyang Z, Zhao B, Liu Z, Zhao Y, Zhang Q (2016) Genetically humanized pigs exclusively expressing human insulin are generated through custom endonuclease-mediated seamless engineering. J Molecular Cell Biol 8(2):174–177

  26. 26.

    Zhou X, Xin J, Fan N, Zou Q, Huang J, Ouyang Z, Zhao Y, Zhao B, Liu Z, Lai S (2015) Generation of CRISPR/Cas9-mediated gene-targeted pigs via somatic cell nuclear transfer. Cell Mol Life Sci 72(6):1175–1184

  27. 27.

    Xin JG, Yang HQ, Fan NN, Zhao BT, Ouyang Z, Liu ZM, Zhao Y, Li XP, Song J, Yang Y et al (2013) Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8(12):9

  28. 28.

    Xin J, Yang H, Fan N, Zhao B, Ouyang Z, Liu Z, Zhao Y, Li X, Song J, Yang Y (2013) Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs. PLoS One 8(12):e84250

  29. 29.

    Bae S, Park J, Kim JS (2014) Cas-OFFinder: a fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30(10):1473–1475

  30. 30.

    Yang D, Xu J, Zhu T, Fan J, Lai L, Zhang J, Chen YE (2014) Effective gene targeting in rabbits using RNA-guided Cas9 nucleases. J Molecular Cell Biol 6(1):97–99

  31. 31.

    Zhang W, Li J, Suzuki K, Qu J, Wang P, Zhou J, Liu X, Ren R, Xu X, Ocampo A (2015) A Werner syndrome stem cell model unveils heterochromatin alterations as a driver of human aging. Science 348(6239):1160–1163

  32. 32.

    Koenig M, Beggs AH, Moyer M, Scherpf S, Heindrich K, Bettecken T, Meng G, Muller CR, Lindlof M, Kaariainen H et al (1989) The molecular-basis for duchenne versus becker muscular-dystrophy—correlation of severity with type of deletion. Am J Hum Genet 45(4):498–506

  33. 33.

    Haghighi K, Kolokathis F, Gramolini AO, Waggoner JR, Pater L, Lynch RA, Fan GC, Tsiapras D, Parekh RR, Dorn GW et al (2006) A mutation in the human phospholamban gene, deleting arginine 14, results in lethal, hereditary cardiomyopathy. Proc Natl Acad Sci USA 103(5):1388–1393

  34. 34.

    Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112(11):3570–3575

  35. 35.

    Qi WW, Zhu T, Tian ZR, Li CB, Zhang W, Song RT (2016) High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BMC Biotechnol 16(1):58

  36. 36.

    Numamoto M, Maekawa H, Kaneko Y (2017) Efficient genome editing by CRISPR/Cas9 with a tRNA-sgRNA fusion in the methylotrophic yeast Ogataea polymorpha. J Biosci Bioeng 124(5): 487–492

  37. 37.

    Hu X, Wang C, Liu Q, Fu Y, Wang K (2017) Targeted mutagenesis in rice using CRISPR-Cpf1 system. J Genet Genom =Yi chuan xue bao 44(1):71–73

  38. 38.

    Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV et al (2016) Crystal structure of Cpf1 in complex with guide RNA and target DNA. Cell 165(4):949–962

  39. 39.

    Mali P, Esvelt KM, Church GM (2013) Cas9 as a versatile tool for engineering biology. Nat Methods 10(10):957

  40. 40.

    Frendewey D, Dingermann T, Cooley L, Söll D (1985) Processing of precursor tRNAs in Drosophila. Processing of the 3′end involves an endonucleolytic cleavage and occurs after 5′end maturation. J Biol Chem 260(1):449–454

  41. 41.

    Ibrahim H, Wilusz J, Wilusz CJ (2008) RNA recognition by 3′-to-5′ exonucleases: the substrate perspective. Biochem Biophys Acta 1779(4):256–265

  42. 42.

    Singh D, Mallon J, Poddar A, Wang Y, Tipanna R, Yang O, Bailey S, Ha T (2017) Real-time observation of DNA target interrogation and product release by the RNA-guided endonuclease CRISPR Cpf1. https://doi.org/10.1101/205575

  43. 43.

    Fan NN, Lai LX (2013) Genetically modified pig models for human diseases. J Genet Genom 40(2):67–73

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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).

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Correspondence to Kepin Wang or Xiaoping Li or Liangxue Lai.

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Wu, H., Liu, Q., Shi, H. et al. Engineering CRISPR/Cpf1 with tRNA promotes genome editing capability in mammalian systems. Cell. Mol. Life Sci. 75, 3593–3607 (2018). https://doi.org/10.1007/s00018-018-2810-3

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Keywords

  • CRISPR/Cpf1
  • gRNAtRNA system
  • Genome editing
  • Rabbit
  • Pig