Crisper - A Gene editing tech.

Crisper cas-9 is a powerful gene editing technology and helps to solve many problems in shorter time and more precisely. The two main molecules is an enzyme CAS-9 and a piece of RNA known as GUIDE RNA, that introduce a change into the DNA.

1. Progress is impossible without change, and those who cannot change their minds cannot change anything George Bernard shaw

2. Gene Editing for Vegetable Crop Disease Resistance Neha Yadav Division of vegetable crops Id no. 10905 2

3. ICAR-Indian Institute of Horticultural Research 3 What is editing??? Addition Deletion Replacement Rewrite What is genome?? Entire genetic material of a organism

4. ICAR-Indian Institute of Horticultural Research 4 Genome editing DNA is inserted , deleted , mutated or replaced at a particular position in the genome of the organism

5. ICAR-Indian Institute of Horticultural Research 5 Rapidly advancing technology Targeted mutations Highly specific and great precision No transgenic modifications Superior to conventional chemical mutagenesis. why gene editing? Georges and Ray, 2017

6. ICAR-Indian Institute of Horticultural Research 6 Why vegetables????? Vegetables and fruits are an important part of a healthy diet. Provides all of the nutrients an excellent source of carbohydrates and aid in disease prevention. can lower blood pressure, reduce risk of heart disease and stroke, prevent some types of cancer, lower risk of eye and digestive problems, and have a positive effect upon blood sugar which can help keep appetite in check.

7. ICAR-Indian Institute of Horticultural Research 7 GENETIC ENGINEERING VS. GENE EDITING Methods of plant genetic improvement

8. ICAR-Indian Institute of Horticultural Research 8 Techniques of genome editing CRISPR/Cas9 ZFN TALEN

9. ICAR-Indian Institute of Horticultural Research 9 Yang et al., 2016

10. ICAR-Indian Institute of Horticultural Research 10 Current status of genome editing in horticultural crops Plant Target genes Technology Traits Reference Solanum lycopersicum ARGONAUTE7 (SlAGO7) CRISPR/Cas9 Leaf development Brooks et al., 2014 SHORT-ROOT (SHR) CRISPR/Cas9 Root development Ron et al., 2014 Ripening inhibitor (RIN) CRISPR/Cas9 Fruit ripening Ali et al., 2015b Anthocyanin 1 (ANT1) CRISPR/Cas9 TALEN Anthocyanin biosynthesis Cermak et al., 2015 Phytoene desaturase ( SlPDS ), Carotenoid biosynthesis Pan et al., 2016 Self pruning 5G (sp5G), self pruning (sp) CRISPR/Cas9 Plant development Soyk et al., 2017 PHYTOENE SYNTHASE (PSY1) Fruit color Hayut et al., 2017 MILDEW RESISTANT LOCUS o (Mlo) CRISPR/Cas9 Powdery mildew resis . Nekrasov et al., 2017 Solanum tuberosum StMYB44 Phosphate transport Zhou et al., 2017 ACETOLACTATE SYNTHASE1 (StALS1) Herbicide resistance Butler et al., 2016 Subburaj et al., 2016

11. ICAR-Indian Institute of Horticultural Research 11 Granule-bound starch synthase (GBSS) Starch quality Andersson et al., 2017 Brassica oleracea Gibberellin3-beta-dioxygenase 1 CRISPR/Cas9 Plant development, fruit dehiscence Lawrenson et al., 2015 Lactuca sativa BRASSINOSTEROID INSENSITIVE 2 (BIN2) CRISPR/Cas9 Plant development Woo et al., 2015 Cucumis sativus Eukaryotic translation initiation factor 4E (eIF4E) CRISPR/Cas9 Virus resistance Chandrasekaran et al., 2016 Watermelon Phytoene desaturase ( ClPDS ) Carotenoid biosynthesis Tian et al., 2017

12. Zinc finger nuclease Zinc Finger Nuclease DNA binding motif Usually present in proteins which binds to DNA Recognize specific DNA sequence Endonuclease Usually DNA cleavage domain of FokI Flavobacterium okeanokoites , is used Non-specific cleavage ICAR-Indian Institute of Horticultural Research Benefits Rapid disruption of, or integration into, any genomic loci Mutations made are permanent and heritable Edits induced through a single transfection experiment Knockout or knock-in cell lines in as little as two months No antibiotic selection required for screening Weeks et al., 2016

13. ICAR-Indian Institute of Horticultural Research 13 TALENs - transcription activator-like effector nucleases TALE Nuclease TALE genes can be mutated to generate sequence-specific DNA binding proteins The modified TALEs can be fused to nucleases for targeted double stranded break

14. ICAR-Indian Institute of Horticultural Research 14 CRISPR as a tool for genome editing Two component system- Guide RNA Endonuclease - Cas9

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17. ICAR-Indian Institute of Horticultural Research 17 Objective : To develop virus resistance in cucumber ( Cucumis sativus L.) using Cas9/ subgenomic RNA ( sgRNA ) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene Chandrasekaran et al., 2016

18. ICAR-Indian Institute of Horticultural Research 18 Plant viruses cause extensive reductions in crop yields worldwide. Several paths to the development of virus resistance in crop plants. One path is classical plant breeding and another path is genome editing

19. ICAR-Indian Institute of Horticultural Research 19 EXPERIMENTAL PROCEDURES

20. ICAR-Indian Institute of Horticultural Research 20 RESULTS Efficacy of CRISPR/Cas9 in the T0 generation : Disrupt eIF4E eIF4E is a plant cellular translation factor essential for the Potyviridae life cycle Natural point mutations in this gene can confer resistance to potyviruses In cucumber, two eIF4E genes have been identified, eIF4E and eIF ( iso )4E

21. ICAR-Indian Institute of Horticultural Research 21 Cas9/sgRNA1 construct was designed to target the sequence in the first exon of eIF4E The Cas9/sgRNA2 construct was designed to target the third exon in the coding region to allow translation of approximately two-thirds of the protein, perhaps without disrupting all of its functions

22. ICAR-Indian Institute of Horticultural Research 22 Fig. 1 Gene editing of eIF4E mediated by CRISPR/Cas9 in transgenic cucumber plants

23. ICAR-Indian Institute of Horticultural Research 23 Genotypes and segregation of T1 mutants of CEC1-1 The T1 progeny segregated into three groups (Fig. 2): Heterozygous plants that contained about equal amounts of undigested and digested DNA (plants 5, 8, 12, 16); Plants with undigested DNA with an intensity stronger than that of digested DNA (plants 2, 9, 7, 20); Non-mutants (wild-type ), with most of the DNA digested (plants 3, 10).

24. ICAR-Indian Institute of Horticultural Research 24 Plant 1 had a 20 nucleotide deletion and plants 4 and 5 had one-nucleotide deletions. Plant 7 had both the 20- and one-nucleotide deletions as observed in T0. Hence, CRISPR/Cas9-induced mutations in cucumber can be stably transmitted through the germ line. Fig. 2 Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1-1 line.

25. ICAR-Indian Institute of Horticultural Research 25 Genotypes and segregation of T1 mutants of CEC1-4 The plant CEC1-4 was self-pollinated and, in the T1 generation, only three of eight plants (Fig. 3, plants 4, 5 and 6) had a faint undigested band on digestion with BmgBI (Fig. 3A). Cloning and sequencing of the undigested faint band showed multiple mutations within the target gene in the same plant.

26. ICAR-Indian Institute of Horticultural Research 26 Fig. 3 Genotyping of eif4e mutants in representative T1 progeny plants of the CEC1-4 line. Polymerase chain reaction (PCR) restriction analysis of Cas9/sgRNA1-mediated mutations (top panel) and transgene insertion (bottom panel) in eight T1 cucumber plants and non-mutant wild-type (wt). (B) Alignment of three eif4e transgenic mutant plants 4, 5and 6 with the wild-type sequence.

27. ICAR-Indian Institute of Horticultural Research 27 Genotypes and segregation of T1 mutants of CEC2-5 Genotyping of CEC2-5 T1 generation plants revealed three groups: homozygous plants with completely undigested DNA (nine of 15 transgenic seedlings) (Fig. 4A, plants 6 and 14); (ii ) Heterozygous plants having similar intensities of undigested and digested DNA (Fig. 4A, plants 9 and 26); (iii) plants in which faintly digested DNA can be seen, which may reflect continuing activity of Cas9/sgRNA2 in heterozygous plants (Fig. 4A, plants 1 and 7)

28. ICAR-Indian Institute of Horticultural Research 28 Fig. 4 Genotyping of the Cas9/sgRNA2-mediated mutation in T1 progeny plants of the CEC2-5 line. Polymerase chain reaction (PCR) restriction analysis of Cas9/ sgRNA2-mediated mutations (top panel) and the presence of the Cas9/sgRNA2 transgene (bottom panel) in eight representative T1 cucumber plants. Alignment of four representative eif4e mutant plants with the wild-type sequence.

29. ICAR-Indian Institute of Horticultural Research 29 Genotypes and segregation of T1 mutants of CEC2-5 To evaluate the types of mutation mediated by Cas9/sgRNA2 in line CEC2-5, the flanking region was PCR amplified and the presence of indel mutations was tested by BglII restriction. The PCR fragment of CEC2-5 was as completely digested as the wild-type. CEC2-5 was cross-pollinated with ‘Bet Alfa’ and the progeny (T1) segregated approximately to 1 : 1 transgenic : non-transgenic. Mutations were observed only in transgenic progeny. Genotyping of CEC2-5 T1 generation plants revealed three groups: ( i ) homozygous plants with completely undigested DNA (nine of 15 transgenic seedlings) (ii) Heterozygous plants having similar intensities of undigested and digested DNA (plants 9 and 26) (iii) plants in which faintly digested DNA can be seen, which may reflect continuing activity of Cas9/sgRNA2 in heterozygous plants (plants 1 and 7)

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31. ICAR-Indian Institute of Horticultural Research 31 Off-target analysis . Cas9/sgRNA1 off-targets were evaluated by the CRISPR-P program using the sgRNA1 sequence against the cucumber genome. Five candidate potential off-targets were determined. PCR and sequencing of these candidate targets revealed no changes in the genome of non-transgenic T3 generation CEC1-1-7-1 Mutations in the putative eIF4E CRISPR/Cas9 sgRNA1 off-target sites

32. ICAR-Indian Institute of Horticultural Research 32 CVYV resistance analysis Virus resistance analysis

33. ICAR-Indian Institute of Horticultural Research 33 ZYMV resistance analysis

34. ICAR-Indian Institute of Horticultural Research 34 Fig. Response of T3 generation plants of non-transgenic CEC-1-7-1 and CEC2-5-M-4n lines to Cucumber vein yellowing virus (CVYV), Zucchini yellow mosaic virus (ZYMV), Papaya ring spot mosaic virus-W (PRSV-W), Cucumber mosaic cucumovirus (CMV) and Cucumber green mottle mosaic tobamovirus (CGMMV) infection at different days post-infection (dpi).

35. ICAR-Indian Institute of Horticultural Research 35 PRSV-W resistance analysis

36. ICAR-Indian Institute of Horticultural Research 36 CONCLUSION CRISPR/Cas9 is an efficient tool for genome editing in cucumber. Disruption of the eIF4E gene in cucumber by CRISPR/Cas9 sgRNA led to the development of virus-resistant plants without otherwise affecting the plant genome. Three generations of backcrossing produced virus resistant plants free of genetic modification , and thus would be considered safe for human consumption and for release into the environment . This novel technology has the potential to expedite the development of pest resistance in many crops without the need for extensive backcrossing and genetic manipulation with wild sources of resistance.

37. ICAR-Indian Institute of Horticultural Research 37 Conclusion As the world population grows, there is an increasing demand for food. This demand needs to be addressed in a sustainable manner e.g. by creating new crop varieties with valuable traits, such as higher yield, enhanced disease resistance , improved salt and drought tolerance . Traditional plant breeding has been used to generate new crop varieties for decades, but new technologies, such as genome editing, have the potential to generate improved varieties faster and at a lower cost, by precise introduction of favorable alleles into many different, locally adapted elite varieties . Genome editing is achieved using sequence-specific nucleases (SSNs) and results in chromosomal changes , such as nucleotide deletions, insertions or substitutions at specified genetic loci . Because certain genetic changes (e.g . nucleotide deletions) introduced using SSNs are indistinguishable from natural mutations, the resulting crop varieties are different from transgenics , aka genetically modified organisms (GMO).

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