Skip to main content
SpringerLink
Log in

CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry

  • Original Article
  • Published:
Plant Cell, Tissue and Organ Culture (PCTOC) Aims and scope Submit manuscript

Abstract

To develop an effective genome editing tool for blueberry breeding, CRISPR-Cas9 and CRISPR-Cas12a were evaluated for their editing efficiencies of a marker gene, beta-glucuronidase (gusA), which was previously introduced into two blueberry cultivars each a single-copy transgene. Four expression vectors were built, with CRISPR-Cas9 and CRISPR-Cas12a each driven by a 35S promoter or AtUbi promoter. Each vector contained two editing sites in the gusA. These four vectors were respectively transformed into the leaf explants of transgenic gusA blueberry and the resulting transgenic calli were induced under hygromycin selection. GUS staining showed that some small proportions of the hygromycin-resistant calli had non-GUS stained sectors, suggesting some possible occurrences of gusA editing. We sequenced GUS amplicons spanning the two editing sites in three blueberry tissues and found about 5.5% amplicons having editing features from the calli transformed with the 35S-Cas9 vector. Further, we conducted a second round of shoot regeneration from leaf explants derived from the initial Cas9- and Cas12a-containing calli (T0) and analyzed amplicons of the target editing region. Of the newly induced shoots, 15.5% for the 35S-Cas9 and 5.3% for the AtUbi-Cas9 showed non-GUS staining, whereas all of the shoots containing the Cas12a vectors showed blue staining. Sanger sequencing confirmed the editing-induced mutations in two representative non-GUS staining lines. Clearly, the second round of regeneration had enriched editing events and enhanced the production of edited shoots. The results and protocol described will be helpful to facilitating high-precision breeding of blueberries using CRISPR Cas technologies.

Key Message

A second round of regeneration enriched editing events and enhanced the production of edited blueberry shoots. The new protocol described facilitates high-precision breeding of blueberries using CRISPR Cas technologies.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

References

  • Alok A, Sandhya D, Jogam P, Rodrigues V, Bhati KK, Sharma H, Kumar J (2020) The rise of the CRISPR/Cpf1 system for efficient genome editing in plants. Front Plant Sci 11

  • Ballington JR (2009) The role of interspecific hybridization in blueberry improvement. Acta Hortic 810:49–59

    Article  Google Scholar 

  • Bandyopadhyay A, Kancharla N, Javalkote VS, Dasgupta S, Brutnell TP (2020) CRISPR-Cas12a (Cpf1): a versatile tool in the plant genome editing tool box for agricultural advancement. Front Plant Sci 11:584151

    Article  PubMed  PubMed Central  Google Scholar 

  • Bibikova M, Golic M, Golic KG, Carroll D (2002) Targeted chromosomal cleavage and mutagenesis in Drosophila using zinc-finger nucleases. Genetics 161:1169–1175

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bortesi L, Fischer R (2015) The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol Adv 33:41–52

    Article  CAS  PubMed  Google Scholar 

  • Brevis P, Bassil N, Ballington J, Hancock J (2008) Impact of wide hybridization on highbush blueberry breeding. J Am Soc Hortic Sci 133:11

    Google Scholar 

  • Charrier A, Vergne E, Dousset N, Richer A, Petiteau A, Chevreau E (2019) Efficient targeted mutagenesis in apple and first time edition of pear using the CRISPR-Cas9 system. Front Plant Sci 10:40

  • Colle M, Leisner CP, Wai CM, Ou S, Bird KA, Wang J, Wisecaver JH, Yocca AE, Alger EI, Tang H, Xiong Z, Callow P, Ben-Zvi G, Brodt A, Baruch K, Swale T, Shiue L, Song GQ, Childs KL, Schilmiller A, Vorsa N, Buell CR, VanBuren R, Jiang N, Edger PP (2019) Haplotype-phased genome and evolution of phytonutrient pathways of tetraploid blueberry. Gigascience 8:giz012

  • Earley KW, Haag JR, Pontes O, Opper K, Juehne T, Song KM, Pikaard CS (2006) Gateway-compatible vectors for plant functional genomics and proteomics. Plant J 45:616–629

    Article  CAS  PubMed  Google Scholar 

  • Ehlenfeldt MK, Prior RL (2001) Oxygen radical absorbance capacity (ORAC) and phenolic and anthocyanin concentrations in fruit and leaf tissues of highbus blueberry. J Agr Food Chem 49:2222–2227

    Article  CAS  Google Scholar 

  • Endo A, Toki S (2019) Targeted mutagenesis using FnCpf1 in Tobacco. Plant Genome Editing with Crispr Systems: Methods and Protocols 1917:269–281

    Article  CAS  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gallardo RK, Zhang Q, Dossett M, Polashock JJ, Rodriguez-Saona C, Vorsa N, Edger PP, Ashrafi H, Babiker E, Finn CE, Iorizzo M (2018) Breeding trait priorities of the blueberry industry in the United States and Canada. HortScience 53:1021

    Article  Google Scholar 

  • Gao J, Wang G, Ma S, Xie X, Wu X, Zhang X, Wu Y, Zhao P, Xia Q (2015) CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Mol Biol 87:99–110

    Article  CAS  PubMed  Google Scholar 

  • Gao W, Long L, Tian X, Xu F, Liu J, Singh PK, Botella JR, Song C (2017) Genome editing in cotton with the CRISPR/Cas9 system. Front Plant Sci 8:1364

    Article  PubMed  PubMed Central  Google Scholar 

  • Goyali JC, Igamberdiev AU, Debnath SC (2018) DNA methylation in lowbush blueberry (Vaccinium angustifolium Ait.) propagated by softwood cutting and tissue culture. Can J Plant Sci 98:1035–1044

    Article  CAS  Google Scholar 

  • Gupta V, Estrada AD, Blakley IC, Reid R, Patel K, Meyer MD, Andersen SU, Brown AF, Lila MA, Loraine A (2015) RNASeq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing. GigaScience 4:5

    Article  PubMed  PubMed Central  Google Scholar 

  • Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J, Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R, Leduc RD, Friedman N, Regev A (2013) De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 8:1494–1512

    Article  CAS  PubMed  Google Scholar 

  • Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) NewAgrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218

    Article  CAS  Google Scholar 

  • Hooghvorst I, Lopez-Cristoffanini C, Nogues S (2019) Efficient knockout of phytoene desaturase gene using CRISPR/Cas9 in melon. Sci Rep 9

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions - beta-glucuronidase as a sensitive and versatile gene fusion marker in higher-plants. Embo J 6:3901–3907

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jeggo PA (1998) DNA breakage and repair. Adv Genet 38:185–218

    Article  CAS  PubMed  Google Scholar 

  • Jia H, Orbovic V, Wang N (2019) CRISPR-LbCas12a-mediated modification of citrus. Plant Biotechnol J 17:1928–1937

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Khanna KK, Jackson SP (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27:247–254

    Article  CAS  PubMed  Google Scholar 

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

  • LeBlanc C, Zhang F, Mendez J, Lozano Y, Chatpar K, Irish VF, Jacob Y (2018) Increased efficiency of targeted mutagenesis by CRISPR/Cas9 in plants using heat stress. Plant J 93:377–386

    Article  CAS  PubMed  Google Scholar 

  • Lee K, Zhang Y, Kleinstiver BP, Guo JA, Aryee MJ, Miller J, Malzahn A, Zarecor S, Lawrence-Dill CJ, Joung JK, Qi Y, Wang K (2019) Activities and specificities of CRISPR/Cas9 and Cas12a nucleases for targeted mutagenesis in maize. Plant Biotechnol J 17:362–372

    Article  CAS  PubMed  Google Scholar 

  • Li B, Rui H, Li Y, Wang Q, Alariqi M, Qin L, Sun L, Ding X, Wang F, Zou J, Wang Y, Yuan D, Zhang X, Jin S (2019) Robust CRISPR/Cpf1 (Cas12a)-mediated genome editing in allotetraploid cotton (Gossypium hirsutum). Plant Biotechnol J 17:1862–1864

    Article  PubMed  PubMed Central  Google Scholar 

  • Lin TY, Walworth A, Zong XJ, Danial GH, Tomaszewski EM, Callow P, Han XM, Zaharia LI, Edger PP, Zhong GY, Song GQ (2019) VcRR2 regulates chilling-mediated flowering through expression of hormone genes in a transgenic blueberry mutant. Hortic Res-England 6

  • Liu HY, Wang K, Jia ZM, Gong Q, Lin ZS, Du LP, Pei XW, Ye XG (2020) Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system. J Exp Bot 71:1337–1349

    Article  CAS  PubMed  Google Scholar 

  • Lowder LG, Zhang D, Baltes NJ, Paul JW 3rd, Tang X, Zheng X, Voytas DF, Hsieh TF, Zhang Y, Qi Y (2015) A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol 169:971–985

    Article  PubMed  PubMed Central  Google Scholar 

  • Mahfouz MM (2017) Genome editing: the efficient tool CRISPR-Cpf1. Nat Plants 3:17028

    Article  CAS  PubMed  Google Scholar 

  • Malzahn AA, Tang X, Lee K, Ren Q, Sretenovic S, Zhang Y, Chen H, Kang M, Bao Y, Zheng X, Deng K, Zhang T, Salcedo V, Wang K, Zhang Y, Qi Y (2019) Application of CRISPR-Cas12a temperature sensitivity for improved genome editing in rice, maize, and Arabidopsis. BMC Biol 17:9

    Article  PubMed  PubMed Central  Google Scholar 

  • Mao YF, Botella JR, Liu YG, Zhu JK (2019) Gene editing in plants: progress and challenges. Natl Sci Rev 6:421–437

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakajima I, Ban Y, Azuma A, Onoue N, Moriguchi T, Yamamoto T, Toki S, Endo M (2017) CRISPR/Cas9-mediated targeted mutagenesis in grape. PLoS ONE 12:e0177966

    Article  PubMed  PubMed Central  Google Scholar 

  • Neto CC (2007) Cranberry and blueberry: evidence for protective effects against cancer and vascular diseases. Mol Nutr Food Res 51:652–664

    Article  CAS  PubMed  Google Scholar 

  • Norris SR, Barrette TR, DellaPenna D (1995) Genetic dissection of carotenoid synthesis in arabidopsis defines plastoquinone as an essential component of phytoene desaturation. Plant Cell 7:11

    Google Scholar 

  • Omori M, Yamane H, Osakabe K, Osakabe Y, Tao R (2021) Targeted mutagenesis of CENTRORADIALIS using CRISPR/Cas9 system through the improvement of genetic transformation efficiency of tetraploid highbush blueberry. J Hortic Sci Biotechnol 96:153–161

    Article  CAS  Google Scholar 

  • Osakabe Y, Liang Z, Ren C, Nishitani C, Osakabe K, Wada M, Komori S, Malnoy M, Velasco R, Poli M, Jung MH, Koo OJ, Viola R, Nagamangala Kanchiswamy C (2018) CRISPR-Cas9-mediated genome editing in apple and grapevine. Nat Protoc 13:2844–2863

    Article  CAS  PubMed  Google Scholar 

  • Park J, Lim K, Kim JS, Bae S (2017) Cas-analyzer: an online tool for assessing genome editing results using NGS data. Bioinformatics 33:286–288

    Article  CAS  PubMed  Google Scholar 

  • Qin G, Gu H, Ma L, Peng Y, Deng XW, Chen Z, Qu LJ (2007) Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis. Cell Res 17:471–482

    Article  CAS  PubMed  Google Scholar 

  • Rowland LJ, Nguyen B (1993) Use of polyethylene-glycol for purification of DNA from leaf tissue of woody-plants. Biotechniques 14:734–740

    CAS  PubMed  Google Scholar 

  • Schindele P, Puchta H (2020) Engineering CRISPR/LbCas12a for highly efficient, temperature-tolerant plant gene editing. Plant Biotechnol J 18:1118–1120

    Article  PubMed  Google Scholar 

  • Shan Q, Wang Y, Li J, Gao C (2014) Genome editing in rice and wheat using the CRISPR/Cas system. Nat Protoc 9:2395–2410

    Article  CAS  PubMed  Google Scholar 

  • Song GQ (2015) Blueberry (Vaccinium corymbosum L.). Methods Mol Biol 1224:121–131

    Article  CAS  PubMed  Google Scholar 

  • Song G-Q, Chen Q (2018) Overexpression of the MADS-box gene K-domain increases the yield potential of blueberry. Plant Sci 276:10

    Article  Google Scholar 

  • Song G, Sink K (2004) Agrobacterium tumefaciens-mediated transformation of blueberry (Vaccinium corymbosum L.). Plant Cell Rep 23:475–484

    Article  CAS  PubMed  Google Scholar 

  • Song GQ, Prieto H, Orbovic V (2019a) Agrobacterium-mediated transformation of tree fruit crops: methods, progress, and challenges. Front Plant Sci 10:226

    Article  PubMed  PubMed Central  Google Scholar 

  • Song GQ, Walworth A, Lin T, Chen Q, Han X, Irina Zaharia L, Zhong GY (2019b) VcFT-induced mobile florigenic signals in transgenic and transgrafted blueberries. Hortic Res 6:105

    Article  PubMed  PubMed Central  Google Scholar 

  • Surridge C (2019) Blueberry fooled into flowering. Nat Plants 5:910–910

    Article  PubMed  Google Scholar 

  • 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 3:17018

    Article  CAS  PubMed  Google Scholar 

  • Vander Kloet SP (1988) The genus Vaccinium in North America. Res Branch Agric Can Publ 1828:201

    Google Scholar 

  • Vu TV, Sivankalyani V, Kim EJ, Doan DTH, Tran MT, Kim J, Sung YW, Park M, Kang YJ, Kim JY (2020) Highly efficient homology-directed repair using CRISPR/Cpf1-geminiviral replicon in tomato. Plant Biotechnol J 18:2133–2143

    Article  CAS  PubMed Central  Google Scholar 

  • Walworth A, Song GQ (2018) The cold-regulated genes of blueberry and their response to overexpression of VcDDF1 in several tissues. Int J Mol Sci 19

  • Wang Z, Wang S, Li D, Zhang Q, Li L, Zhong C, Liu Y, Huang H (2018) Optimized paired-sgRNA/Cas9 cloning and expression cassette triggers high-efficiency multiplex genome editing in kiwifruit. Plant Biotechnol J 16:1424–1433

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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 15:713–717

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin X, Biswal AK, Dionora J, Perdigon KM, Balahadia CP, Mazumdar S, Chater C, Lin HC, Coe RA, Kretzschmar T, Gray JE, Quick PW, Bandyopadhyay A (2017) CRISPR-Cas9 and CRISPR-Cpf1 mediated targeting of a stomatal developmental gene EPFL9 in rice. Plant Cell Rep 36:745–757

    Article  CAS  PubMed  Google Scholar 

  • Zaidi SS, Mahfouz MM, Mansoor S (2017) CRISPR-Cpf1: a new tool for plant genome editing. Trends Plant Sci 22:550–553

    Article  CAS  PubMed  Google Scholar 

  • 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 163:759–771

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zetsche B, Heidenreich M, Mohanraju P, Fedorova I, Kneppers J, DeGennaro EM, Winblad N, Choudhury SR, Abudayyeh OO, Gootenberg JS, Wu WY, Scott DA, Severinov K, van der Oost J, Zhang F (2017) Multiplex gene editing by CRISPR-Cpf1 using a single crRNA array. Nat Biotechnol 35:31–34

    Article  CAS  PubMed  Google Scholar 

  • Zhong ZH, Zhang YX, You Q, Tang X, Ren QR, Liu SS, Yang LJ, Wang Y, Liu XP, Liu BL, Zhang T, Zheng XL, Le Y, Zhang Y, Qi YP (2018) Plant genome editing using FnCpf1 and LbCpf1 nucleases at redefined and altered PAM sites. Mol Plant 11:999–1002

    Article  CAS  PubMed  Google Scholar 

Download references

Funding

This research was supported in part by the agreement of Non-Assistance Cooperative Agreement #58-8060-6-009 between the USDA Agricultural Research Service and Michigan State University. Dr. Xiaoyan Han’s visit at Michigan State University was partially supported by a CAS Scholarship. Dr. Emadeldin A. H. Ahmed is a Fulbright fellow at Michigan State University.

Author information

Author notes

  1. Xiaoyan Han and Yingzhen Yang have contributed equally.

Authors and Affiliations

  1. Plant Biotechnology Resource and Outreach Center, Department of Horticulture, Michigan State University, East Lansing, MI, 48824, USA

    Xiaoyan Han, Xue Han, John T. Ryner, Emadeldin A. H. Ahmed & Guo-qing Song

  2. Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences, Beijing, 100093, People’s Republic of China

    Xiaoyan Han

  3. Grape Genetics Research Unit, USDA-Agricultural Research Service, Geneva, NY, 14456, USA

    Yingzhen Yang & Gan-yuan Zhong

  4. Breeding Research Department of Fruit Trees, Ornamental and Woody Plants, Horticulture Research Institute, Agricultural Research Center, Giza, Egypt

    Emadeldin A. H. Ahmed

  5. Department of Plant Science and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA

    Yiping Qi

Authors
  1. Xiaoyan Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yingzhen Yang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Xue Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. John T. Ryner
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Emadeldin A. H. Ahmed
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Yiping Qi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gan-yuan Zhong
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Guo-qing Song
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

GS and GZ conceived and supervised the research. YY, XYH, XH, JR, EA, and GS conducted the experiments. YY, XYH, and YQ analyzed the data. GS, XYH, YY, YQ, and GZ wrote the manuscript. All authors reviewed and approved the final manuscript.

Corresponding authors

Correspondence to Gan-yuan Zhong or Guo-qing Song.

Ethics declarations

Conflict of interest

The authors declare that no competing interests exist.

Additional information

Communicated by Joyce Van Eck.

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Han, X., Yang, Y., Han, X. et al. CRISPR Cas9- and Cas12a-mediated gusA editing in transgenic blueberry. Plant Cell Tiss Organ Cult 148, 217–229 (2022). https://doi.org/10.1007/s11240-021-02177-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11240-021-02177-1

Keywords

Search

Navigation