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Functional & Integrative Genomics

, Volume 18, Issue 1, pp 89–99 | Cite as

CRISPR/Cas9-mediated efficient editing in phytoene desaturase (PDS) demonstrates precise manipulation in banana cv. Rasthali genome

  • Navneet Kaur
  • Anshu Alok
  • Shivani
  • Navjot Kaur
  • Pankaj Pandey
  • Praveen Awasthi
  • Siddharth Tiwari
Original Article

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has been reported for precise genome modification in many plants. In the current study, we demonstrate a successful mutation in phytoene desaturase (RAS-PDS) of banana cv. Rasthali using the CRISPR/Cas9 system. Two PDS genes were isolated from Rasthali (RAS-PDS1 and RAS-PDS2), and their protein sequence analysis confirmed that both PDS comprises conserved motifs for enzyme activity. Phylogenetic analysis of RAS-PDS1 and RAS-PDS2 revealed a close evolutionary relationship with other monocot species. The tissue-specific expression profile of RAS-PDS1 and RAS-PDS2 in Rasthali suggested differential regulation of the genes. A single 19-bp guide RNA (gRNA) was designed to target the conserved region of these two RAS-PDS and transformed with Cas9 in embryogenic cell suspension (ECS) cultures of cv. Rasthali. Complete albino and variegated phenotype were observed among regenerated plantlets. DNA sequencing of 13 plants confirmed the indels with 59% mutation frequency in RAS-PDS, suggesting activation of the non-homologous end-joining (NHEJ) pathway. The majority of mutations were either insertion (1–5) or deletion (1–4) of nucleotides near to protospacer adjacent motif (PAM). These mutations have created stop codons in RAS-PDS sequences which suggest premature termination of RAS-PDS protein synthesis. The decreased chlorophyll and total carotenoid contents were detected in mutant lines that revealed the functional disruption of both RAS-PDS genes. Our results demonstrate that genome editing through CRISPR/Cas9 can be applied as an efficient tool for banana genome modification.

Keywords

Banana transformation Carotenoid Embryonic cell suspension Genome editing Mutation 

Notes

Acknowledgements

The authors are thankful to the Biotechnology Industry Research Assistance Council (BIRAC) for the banana biofortification project grant to ST. S and NK are thankful to the Department of Biotechnology, Panjab University, Chandigarh, for Ph.D. registration. The authors would like to acknowledge the DBT-eLibrary Consortium (Del-CON) for providing access to online journals.

Funding information

The research was funded by National Agri-Food Biotechnology Institute (NABI), Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10142_2017_577_Fig8_ESM.gif (329 kb)
Fig. S1

Different in-vitro stages of Rasthali. (a) Somatic embryos emerged on embryogenic callus. (b) Embryonic Cell Suspension (ECS). (c) Microscopic view of ECS showing dense cytoplasm and spherical structure. (d) Transformed ECS on embryo regeneration selection medium. (GIF 328 kb)

10142_2017_577_MOESM1_ESM.tif (7.4 mb)
High Resolution Image (TIFF 7583 kb)
10142_2017_577_Fig9_ESM.gif (121 kb)
Fig. S2

Gel image of amplified ~1468 bp fragment from genomic DNA of transformed and control plants. Lines (L-8 to L-64) represent putative mutant plants, C is control (empty vector plant) and N represent negative control (non-transgenic plant). (GIF 121 kb)

10142_2017_577_MOESM2_ESM.tif (2.8 mb)
High Resolution Image (TIFF 2832 kb)
10142_2017_577_Fig10_ESM.gif (121 kb)
Fig. S3

Gel image of amplified 311 bp fragment from genomic DNA of transformed and control plants. Lines (L-8 to L-64) represent putative mutant plants, C is control (empty vector plant) and N represents negative control (non-template control). The ladder represents 1 kb ladder of GeneRular. (GIF 121 kb)

10142_2017_577_MOESM3_ESM.tif (2.4 mb)
High Resolution Image (TIFF 2484 kb)
10142_2017_577_Fig11_ESM.gif (100 kb)
Fig. S4

Chromatogram. With respect to control (a), results show deletion (b) and insertion (c, d) in the sequence. Red marked nucleotides show mutation. In control sequence, insertions are represented with capital letters (Lines 16 and 41) and deletions are represented by small letters (Line 08). (GIF 99.5 kb)

10142_2017_577_MOESM4_ESM.tif (4.5 mb)
High Resolution Image (TIFF 4646 kb)
10142_2017_577_Fig12_ESM.gif (504 kb)
Fig. S5

Editing in different RAS-PDS sequence leads to the formation of the stop codon. (GIF 503 kb)

10142_2017_577_MOESM5_ESM.tif (22.9 mb)
High Resolution Image (TIFF 23419 kb)
10142_2017_577_Fig13_ESM.gif (35 kb)
Fig. S6

Absorption spectra at 470, 653 and 665 for control (empty vector plant) and mutant lines. (GIF 34.8 kb)

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High Resolution Image (TIFF 1385 kb)
10142_2017_577_MOESM7_ESM.docx (16 kb)
Table S1 (DOCX 15.5 kb)

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Copyright information

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Authors and Affiliations

  1. 1.National Agri-Food Biotechnology Institute (NABI), Department of BiotechnologyMinistry of Science and Technology (Government of India)MohaliIndia
  2. 2.Department of BiotechnologyPanjab UniversityChandigarhIndia

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