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Application of Cas12a and nCas9-activation-induced cytidine deaminase for genome editing and as a non-sexual strategy to generate homozygous/multiplex edited plants in the allotetraploid genome of tobacco

  • Chen-Tran Hsu
  • Yu-Jung Cheng
  • Yu-Hsua Yuan
  • Wei-Fon Hung
  • Qiao-Wei Cheng
  • Fu-Hui Wu
  • Lan-Ying Lee
  • Stanton B. Gelvin
  • Choun-Sea LinEmail author
Article

Key message

Protoplasts can be used for genome editing using several different CRISPR systems, either separately or simultaneously, and that the resulting mutations can be recovered in regenerated non-chimaeric plants.

Abstract

Protoplast transfection and regeneration systems are useful platforms for CRISPR/Cas mutagenesis and genome editing. In this study, we demonstrate the use of Cpf1 (Cas12a) and nCas9-activation-induced cytidine deaminase (nCas9-Target-AID) systems to mutagenize Nicotiana tabacum protoplasts and to regenerate plants harboring the resulting mutations. We analyzed 20 progeny plants of Cas12a-mediated phytoene desaturase (PDS) mutagenized regenerants, as well as regenerants from wild-type protoplasts, and confirmed that their genotypes were inherited in a Mendelian manner. We used a Cas9 nickase (nCas9)-cytidine deaminase to conduct C to T editing of the Ethylene receptor 1 (ETR1) gene in tobacco protoplasts and obtained edited regenerates. It is difficult to obtain homozygous edits of polyploid genomes when the editing efficiency is low. A second round of mutagenesis of partially edited regenerants (a two-step transfection protocol) allowed us to derive ETR1 fully edited regenerants without the need for sexual reproduction. We applied three different Cas systems (SaCas9, Cas12a, and nCas9-Traget AID) using either a one-step or a two-step transfection platform to obtain triply mutated and/or edited tobacco regenerants. Our results indicate that these three Cas systems can function simultaneously within a single cell.

Keywords

CRISPR/Cas Chimeric plants Cas12a Cytidine deaminase Polyploid genome editing 

Notes

Acknowledgements

We acknowledge Drs. Masaki Endo and Seiichi Toki of the Plant Genome Engineering Research Unit, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, for Cas12a plasmids. This research was supported by Academia Sinica, Innovative Translational Agricultural Research Administrative Office (AS-KPQ-107-ITAR-10), and the Ministry of Science and Technology (105-2313-B-001 -007 -MY3), Taiwan.

Author contributions

All authors conceived and designed the experiments. CSL, CTH, YJC, QWC, YHY, and WFH performed targeted mutagenesis analysis. CTH and CSL conducted protoplast regeneration. CTH, YJC, QWC, YHY, QWC, FHW, and WFH performed molecular biology experiments. CSL, LYL, and SBG interpreted the data. CSL, LYL, and SBG wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

11103_2019_907_MOESM1_ESM.pdf (22.8 mb)
Supplementary material 1 (PDF 23310 kb)

References

  1. Andersson M, Turesson H, Nicolia A, Fält AS, Samuelsson M, Hofvander P (2017) Efficient targeted multiallelic mutagenesis in tetraploid potato (Solanum tuberosum) by transient CRISPR-Cas9 expression in protoplasts. Plant Cell Rep 36:117–128CrossRefGoogle Scholar
  2. Andersson M, Turesson H, Olsson N, Fält AS, Ohlsson P, Gonzalez MN, Samuelsson M, Hofvander P (2018) Genome editing in potato via CRISPR-Cas9 ribonucleoprotein delivery. Physiol Plant 164:378–384CrossRefGoogle Scholar
  3. Cao WH, Liu J, He XJ, Mu RL, Zhou HL, Chen SY, Zhang JS (2007) Modulation of ethylene responses affects plant salt-stress responses. Plant Physiol 143:707–719CrossRefGoogle Scholar
  4. Collier R, Thomson JG, Thilmony R (2018) A versatile and robust Agrobacterium-based gene stacking system generates high-quality transgenic Arabidopsis plants. Plant J 95:573–583CrossRefGoogle Scholar
  5. Endo A, Masafumi M, Kaya H, Toki S (2016) Efficient targeted mutagenesis of rice and tobacco genomes using Cpf1 from Francisella novicida. Sci Rep 6:38169CrossRefGoogle Scholar
  6. Fossi M, Amundson K, Kuppu S, Britt A, Comai L (2019) Regeneration of Solanum tuberosum plants from protoplasts induces widespread genome instability. Plant Physiol 180:78–86CrossRefGoogle Scholar
  7. Gao L, Cox DBT, Yan WX, Manteiga JC, Schneider MW, Yamano T, Nishimasu H, Nureki O, Crosetto N, Zhang F (2017) Engineered Cpf1 variants with altered PAM specificities. Nat Biotechnol 35:789–792CrossRefGoogle Scholar
  8. Hall AE, Bleecker AB (2003) Analysis of combinatorial loss-of-function mutants in the Arabidopsis ethylene receptors reveals that the ers1 etr1 double mutant has severe developmental defects that are EIN2 dependent. Plant Cell 15:2032–2041CrossRefGoogle Scholar
  9. Hill JT, Demarest BL, Bisgrove BW, Su YC, Smith M, Yost HJ (2014) Poly peak parser: method and software for identification of unknown indels using Sanger sequencing of PCR products. Dev Dyn 243:1632–1636CrossRefGoogle Scholar
  10. Hu X, Wang C, Liu Q, Fu Y, Wang K (2017) Targeted mutagenesis in rice using CRISPR-Cpf1 system. J Genet Genom 44:71–73CrossRefGoogle Scholar
  11. Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR (2018) Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556:57–63CrossRefGoogle Scholar
  12. 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 337:816–821CrossRefGoogle Scholar
  13. Kaya H, Mikami M, Endo A, Endo M, Toki S (2016) Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9. Sci Rep 6:26871CrossRefGoogle Scholar
  14. Kim H, Kim ST, Ryu J, Kang BC, Kim JS, Kim SG (2017) CRISPR/Cpf1-mediated DNA-free plant genome editing. Nat Commun 8:14406CrossRefGoogle Scholar
  15. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JR, Aryee MJ, Joung JK (2015) Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523:481–485CrossRefGoogle Scholar
  16. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424CrossRefGoogle Scholar
  17. Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, Kim YB, Badran AH, Liu DR (2017) Improved base excision repair inhibition and bacteriophage Mu Gam protein yields C:G-to-T: a base editors with higher efficiency and product purity. Sci Adv 3(8):eaao4774CrossRefGoogle Scholar
  18. Lee L-Y, Fang M-J, Kuang L-Y, Gelvin SB (2008) Vectors for multi-color bimolecular fluorescence complementation to investigate protein-protein interactions in living plant cells. Plant Meth 4:24CrossRefGoogle Scholar
  19. Lee L-Y, Wu F-H, Hsu C-T, Shen S-C, Yeh H-Y, Liao D-C, Fang M-J, Liu N-T, Yen Y-C, Dokládal L, Sykorová E, Gelvin SB, Lin CS (2012) Screening a cDNA library for protein-protein interactions directly in planta. Plant Cell 24:1746–1759CrossRefGoogle Scholar
  20. Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J (2013) Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol 31:688–691CrossRefGoogle Scholar
  21. Li J, Sun Y, Du J, Zhao Y, Xia L (2017) Generation of targeted point mutations in rice by a modified CRISPR/Cas9 system. Mol Plant 10:526–529CrossRefGoogle Scholar
  22. Lin C-S, Hsu C-T, Yang L-H, Lee L-Y, Fu J-Y, Cheng Q-W, Wu F-H, Hsiao H-C, Zhang Y, Zhang R, Chang W-J, Yu C-T, Wang W, Liao L-J, Gelvin SB, Shih MC (2018) Application of protoplast technology to CRISPR/Cas9 mutagenesis: from single-cell mutation detection to mutant plant regeneration. Plant Biotechnol J 16:1295–1310CrossRefGoogle Scholar
  23. Lu Y, Zhu JK (2017) Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system. Mol Plant 10:523–525CrossRefGoogle Scholar
  24. Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG (2015) A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants. Mol Plant 8:1274–1284CrossRefGoogle Scholar
  25. Marx V (2016) Plants: a tool box of cell-based assays. Nat Meth 13:551–554CrossRefGoogle Scholar
  26. Medgyesy P, Menczel L, Maliga P (1980) The use of cytoplasmic streptomycin resistance: chloroplast transfer from Nicotiana tabacum into Nicotiana sylvestris, and isolation of their somatic hybrids. Mol Gen Genet 179:693–698CrossRefGoogle Scholar
  27. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  28. Nekrasov V, Staskawicz B, Weigel D, Jones JD, Kamoun S (2013) Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nat Biotechnol 31:691–693CrossRefGoogle Scholar
  29. Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A (2016) Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353:aaf8729CrossRefGoogle Scholar
  30. O’Malley RC, Rodriguez FI, Esch JJ, Binder BM, O’Donnell P, Klee HJ, Bleecker AB (2005) Ethylene-binding activity, gene expression levels, and receptor system output for ethylene receptor family members from Arabidopsis and tomato. Plant J 41:651–659CrossRefGoogle Scholar
  31. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA (2013) Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152:1173–1183CrossRefGoogle Scholar
  32. Qu X, Hall BP, Gao Z, Schaller GE (2007) A strong constitutive ethylene-response phenotype conferred on Arabidopsis plants containing null mutations in the ethylene receptors ETR1 and ERS1. BMC Plant Biol 7:3CrossRefGoogle Scholar
  33. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F (2015) In vivo genome editing using Staphylococcus aureus Cas9. Nature 520:186–191CrossRefGoogle Scholar
  34. Ren B, Yan F, Kuang Y, Li N, Zhang D, Lin H, Zhou H (2017) A CRISPR/Cas9 toolkit for efficient targeted base editing to induce genetic variations in rice. Sci China Life Sci 60:516–519CrossRefGoogle Scholar
  35. Ren B, Yan F, Kuang Y, Li N, Zhang D, Zhou X, Lin H, Zhou H (2018) Improved base editor for efficiently inducing genetic variations in rice with CRISPR/Cas9-guided hyperactive hAID mutant. Mol Plant 11:623–626CrossRefGoogle Scholar
  36. Shan Q, Wang Y, Li J, Zhang Y, Chen K, Liang Z, Zhang K, Liu J, Xi JJ, Qiu JL, Gao C (2013) Targeted genome modification of crop plants using a CRISPR-Cas system. Nat Biotechnol 31:686–688CrossRefGoogle Scholar
  37. Sheu JJ, Yu TS, Tong WF, Yu SM (1996) Carbohydrate starvation stimulates differential expression of rice α-amylase genes that is modulated through complicated transcriptional and postranscriptional process. J Biol Chem 27:26998–27004CrossRefGoogle Scholar
  38. Shimatani Z, Kashojiya S, Takayama M, Terada R, Arazoe T, Ishii H, Teramura H, Yamamoto T, Komatsu H, Miura K, Ezura H, Nishida K, Ariizumi T, Kondo A (2017) Targeted base editing in rice and tomato using a CRISPR-Cas9 cytidine deaminase fusion. Nat Biotechnol 35:441–443CrossRefGoogle Scholar
  39. Tang X, Lowder LG, Zhang T, Malzahn AA, Zheng X, Voytas DF, Zhong Z, Chen Y, Ren Q, Li Q, Kirkland ER, Zhang Y, Qi Y (2017) A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants. Nat Plants 3:17018CrossRefGoogle Scholar
  40. Vojta A, Dobrinić P, Tadić V, Bočkor L, Korać P, Julg B, Klasić M, Zoldoš V (2016) Repurposing the CRISPR-Cas9 system for targeted DNA methylation. Nucl Acids Res 44:5615–5628CrossRefGoogle Scholar
  41. Wang M, Mao Y, Lu Y, Tao X, Zhu JK (2017) Multiplex gene editing in rice using the CRISPR-Cpf1 system. Mol Plant 10:1011–1013CrossRefGoogle Scholar
  42. Woo JW, Kim J, Kwon SI, Corvalán C, Cho SW, Kim H, Kim SG, Kim ST, Choe S, Kim JS (2015) DNA-free genome editing in plants with preassembled CRISPR-Cas9 ribonucleoproteins. Nat Biotechnol 33:1162–1164CrossRefGoogle Scholar
  43. Xie K, Minkenberg B, Yang Y (2015) Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci USA 112:3570–5357CrossRefGoogle Scholar
  44. Xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang XC, Chen QJ (2014) A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol 14:327CrossRefGoogle Scholar
  45. 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 15:713–717CrossRefGoogle Scholar
  46. Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771CrossRefGoogle Scholar
  47. Zhong Z, Zhang Y, You Q, Tang X, Ren Q, Liu S, Yang L, Wang Y, Liu X, Liu B, Zhang T, Zheng X, Le Y, Zhang Y, Qi Y (2018) Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol Plant 11:999–1002CrossRefGoogle Scholar
  48. Zong Y, Wang Y, Li C, Zhang R, Chen K, Ran Y, Qiu JL, Wang D, Gao C (2017) Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion. Nat Biotechnol 35:438–440CrossRefGoogle Scholar
  49. Zong Y, Song Q, Li C, Jin S, Zhang D, Wang Y, Qiu JL, Gao C (2018) Efficient C-to-T base editing in plants using a fusion of nCas9 and human APOBEC3A. Nat Biotechnol 36:950–953CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Agricultural Biotechnology Research Center, Academia SinicaTaipeiTaiwan
  2. 2.Department of Biological SciencesPurdue UniversityWest LafayetteUSA

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