Genome editing

Student purpose...... with its application in agriculture

1. Sudeep Pandey PALB 6306 Department of Plant Pathology

2. GENOME  A  genome  is the  genetic material  of an organism. It consists of DNA  (or  RNA in RNA viruses). The genome includes both the  genes  (the  coding regions), the  noncoding DNA  and the genetic material of the  mitochondria  and chloroplasts . The term  genome  was created in 1920 by  Hans Winkler ,  professor of botany at the University of Hamburg, Germany.

3. GENOME EDITING the concept of genome editing is not limited to genes. also includes making alterations to non-coding regions of genomes and to epigenomes . Targeted interventions Alter the structural or functional characteristics like colour or number of blooms in flowering plants some disease traits in animals and plants

4. GENOME EDITING This approach is called  reverse genetics   Among the key requirements of reverse genetic analysis is the ability to modify the DNA sequence of the target organisms. Genome editing was selected by  Nature Methods   as the 2011 Method of the Year . The CRISPR- Cas system was selected by as 2015 Breakthrough of the Year

5. Pathogens weapon Can be neutralized by Pathogen produce virulence factor Suppression of Virulence Functions Plants have susceptibility gene Genome editing Many pathogens attack crop Introduction of novel R genes and stacking multiple genes at a preferred site. Pathogen has defeated many R gene Restore defeated R genes Ways to produce disease resistance plant (Sebastian, 2013)

6. Methods for plant genome manipulation Classical breeding Transgenic approach Targeted genome editing An alternative to both classical plant breeding and transgenic approach

7. Comparison between traditional and modern genome editing technologies Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-ray or fast neutron radiation) Biological (ZFNs, TALENs or CRISPR/ Cas ) Biological - Transgenics (e.g., Agro or gene gun) Characteristics of genetic variation Substitution and Deletion Deletion and chromosomal mutation Substitution and Deletion and insertion Insertions Loss of function Loss of function Loss of function and gain of function Loss of function and gain of function Advantages Not necessary of knowing gene function or sequences Not necessary of knowing gene function or sequences Gene specific mutation Insertion of genes of known functions into host plant genome Easy production of random mutation Easy production of random mutation Efficient production of desirable mutation Efficient creation of plants with desirable traits 9

8. Disadvantages Inefficient screening of desirable traits Inefficient screening of desirable traits Necessity of knowing gene function and sequences Necessity of knowing gene function and sequences Non specific mutation Non specific mutation Prerequisite of efficient genetic transformation Prerequisite of efficient genetic transformation Other features Non transgenic process and traits Non transgenic process and traits Transgenic process but non transgenic traits Transgenic process and traits 10 Mutagen Chemical(e.g., EMS) Physical (e.g., gamma, X-ray or fast neutron radiation) Biological (ZFNs, TALENs or CRISPR/ Cas ) Biological - Transgenics (e.g., Agro or gene gun)

9. Introduction… Genome editing , or  genome editing with engineered nucleases  ( GEEN ) is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, or "molecular scissors”.   The nucleases create specific double-strand breaks (DSBs) at desired locations in the genome and harness the cell’s endogenous mechanisms to repair the induced break by natural processes of  homologous recombination  (HR) and  non-homologous end-joining  (NHEJ). 11

10. Non-homologous end-joining (NHEJ ) Homologous recombination (HR) Rejoins the broken ends and is often accompanied by loss/gain of some nucleotides Repair DNA as a template to restore the DSBs Thus the outcome of NHEJ is variable Outcome of this kind of repair is precise and controllable ( Hyongbum , 2014)

11. Why genome editing? To understand the function of a gene or a protein, one interferes with it in a sequence-specific way and monitors its effects on the organism. In some organisms, it is difficult or impossible to perform site-specific mutagenesis , and therefore more indirect methods must be used, such as silencing the gene of interest by short RNA interference ( siRNA ). But sometime gene disruption by  siRNA  can be variable or incomplete. Nucleases such as CRISPR can cut any targeted position in the genome and introduce a modification of the endogenous sequences for genes that are impossible to specifically target using conventional  RNAi . 13

12. Requirement : A homing device: for specific identification of target sequence An endonuclease: for creating double strand break Uses : Gene knock out Gene tagging Specific mutation (insertion/deletion study) Gene knock in Promoter study 5

13. Novel tools for genome editing Zinc finger nuclease (ZFN) TALENs CRISPER/Cas9 Meganuclease 6 Transcription activator like effector nucleases Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR associated (Cas) protein

14. 1. Mega Nuclease First tool used for double strand break-induced genome manipulation Occur naturally in Yeast and in Chlamydomonas In these enzymes binding site and restriction site occur within same unit hence difficult to modify Crop where it is used Crop/plant Trait Reference Maize Herbicide resistance Gao et al , 2010 Cotton Herbicide resistance Insect resistance D’Halluin et al., 2013 Limitation -Difficult to manipulate the DNA binding site -Small recognition site

15. 2. Zinc finger nuclease Zinc finger protein They were first identified as a DNA-binding motif in  Transcription factor  TFIIIA from  African clawed frog ( Xenopus   laevis ) Small protein structural motif that is characterized by the coordination of one or more  zinc ions  in order to stabilize the fold contain multiple finger-like protrusions that make tandem contacts with their target molecule These are hybrid restriction enzymes Zn H H C C Consist of two parts:  generated by fusing a  zinc finger DNA-binding domain  to a  DNA-cleavage domain

16.   FokI , naturally found in   Flavobacterium   okeanokoites N-terminal binding domain  and a non-specific DNA cleavage domain at the C- terminal Fok1

17. DEVELOPMENT OF ZINC FINGER NUCLEASE Zinc finger domains can be engineered to target specific desired  DNA   sequences and this enables zinc-finger nucleases to target unique sequences within complex  genomes . By taking advantage of endogenous DNA repair machinery , these reagents can be used to precisely alter the genomes of higher organisms .

18. Mode of action 1.Binding of ZFN to DNA 2.Restricting the DNA 3.Cut sequence may be deleted/new sequence may be added 4.Break end will be sealed by host own repairing mechanism 5’ 3’ 3’ 5’ + -

19. Crop where it was used Crop/plant Trait Rererence Maize Herbicide tolerance Shukla et al. , 2009 Soybean Physiological trait Curtin et al., 2011 Tomato against TYLCV Takenaka et al., 2007 12 Limitation Off target effect Construction is cumbersome and time consuming

20. 3. Transcription activator like effector nucleases (TALENs) First time reported by Ulla Bonas in Xanthomonas oryzae (1989) Bacterial cell Consist of TALE + Endonuclease Prof. Ulla Bonas Plant cell Nucleus Effector Divert metabolic machinery of host towards the pathogen 15

21. TAL  ( transcription activator-like )  effectors are  proteins secreted by  Xanthomonas  bacteria via their  type III secretion system when they infect various  plant  species. These proteins can bind  promoter  sequences in the host plant and activate the  expression  of plant genes that aid bacterial infection. Transcription activator-like effector nucleases  (TALEN) are  restriction enzymes  that can be engineered to cut specific sequences of DNA. They are made by fusing a  TAL effector   DNA-binding domain to a DNA cleavage domain (a  nuclease  which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind practically any desired DNA sequence, so when combined with a nuclease, DNA can be cut at specific locations. 16

22. An N-terminal domain containing a type III secretion signal A central repeat domain that determines DNA-binding specificity TALEs are organized into three sections a C-terminal domain containing a nuclear localization signal and an acidic activation domain A stretch of 34 amino acid repeated at 15.5 - 19.5 times ……… ……… 34 amino acid 12 Repeat variable diresidues (RVD) 13 In each repeat amino acid at the position 12 and 13 varies thus form a Repeat variable diresidues(RVDs) The amino acid identity of the RVDs is responsible for DNA nucleotide recognition, enabling the design of TALENs to target unique DNA sequences Molecular structure of effector

23. Once a DNA target is identified, an RVD for each target base is selected according to the following code: Designing TALEN G NN, NH, NK DNA base Amino acid in TALE C HD T NG A NI Bio- informatic tools available for predicting Binding site Programme Website Refernece Target Finder ( https://tale-nt.cac.cornell.edu /) Doyle et al .,  2012 Talvez ( http://bioinfo.mpl.ird.fr/cgibin/talvez/talvez.cgi ) Pérez-Quintero et al .,  2013 Storyteller (http://bioinfoprod.mpl.ird.fr/xantho/tales ) Pérez-Quintero et al.,  2013 ( Mussolino and Cathomen , 2012)

24. Mode of action Fok1 Fok1 Fok1 5’ 3’ 3’ 5’ Fok1 + _ 1. Binding of TALEN 2. Cutting at target site 3 In/del 5’ 3’ 3’ 5’ 4. Gap sealing

25. Different ways by TALENs can be use for Disease resistance plant Transcription activation of different R gene Transcription Repression of different Susceptibility Gene Mutation in Promoters of Susceptibility Genes Gene replacement Destroying pathogen genome (Sebastian,2013)

26. Examples of targeted gene modification using TALENs in plants (Yong Zhang, 2013)

27. CRISPR – Cas systems These are the part of the Bacterial immune system which detects and recognize the foreign DNA and cleaves it. THE CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci Cas (CRISPR- associated) proteins can target and cleave invading DNA in a sequence – specific manner. A CRISPR array is composed of a series of repeats interspaced by spacer sequences acquired from invading genomes. 29

28. HISTORY

29. Components of CRISPR Protospacer adjacent motif  ( PAM ) CRISPR-RNA ( crRNA ) trans-activating crRNA  ( tracrRNA )

30. Different Cas proteins and their f unction Protein Distribution Process Function Cas1 Universal   Spacer acquisition DNAse , not sequence specfic , can bind RNA; present in all Types Cas2 Universal   Spacer acquisition specific to U-rich regions; present in all Types   Cas3 Type I signature   Target interference DNA helicase, endonuclease Cas4 Type I, II   Spacer acquisition RecB -like nuclease with exonuclease activity homologous to RecB Cas5 Type I   crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas6 Type I, III   crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas7 Type I   crRNA expression RAMP protein, endoribonuclease involved in crRNA biogenesis; part of CASCADE Cas8 Type I   crRNA expression Large protein with McrA /HNH-nuclease domain and RuvC -like nuclease; part of CASCADE Cas9 Type II signature   Target interference Large multidomain protein with McrA -HNH nuclease domain and RuvC -like nuclease domain; necessary for interference and target cleavage Cas10 Type III signature   crRNA expression and interference   HD nuclease domain, palm domain, Zn ribbon; some homologies with CASCADE elements 32

31. Protospacer adjacent motif  ( PAM ) is a DNA sequence immediately following the DNA sequence targeted by the  Cas9 nuclease  in the  CRISPR  bacterial  adaptive immune system . PAM is a component of the invading virus or plasmid, but is not a component of the bacterial  CRISPR locus . Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence . PAM is an essential targeting component (not found in bacteria) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease

32. 34 Structure of crRNA

33. Trans-activating crRNA (Tracr) RNA A  trans -encoded small RNA with 24 nucleotide complementarity to the repeat regions of crRNA precursor transcripts Function Pair with Cr RNA for its maturation by processing through RNAseIII Activating Cr RNA-guided cleavage by cas 9 (Jinek, 2012)

34. Action of CRISPR in bacteria The CRISPR immune system works to protect bacteria from repeated viral attack via three basic steps: Adaptation (2) Production of cr RNA (3) Targeting 36

35. 37

36. Versatile Nature of CRISPR Technology 38 Jeffry et al ., 2014

37. On line designing tools Software Work ZiFit (http://zifit.partners.org/ZiFiT/) Helps to construct gRNAs , TALENs, and ZFNs targeting the sequence of interest CRISPR designing tools (http://crispr.mit.edu/) Helps design gRNA sequences that are predicted to minimize off-target mutations E-CRISP (http://e-crisp-test.dkfz.de/E-CRISP/ index.html) Permits the finding of paired gRNAs and off targets CRISPR-PLANT Database (http://www.genome.arizona.edu/crispr/index.html) An online tool that includes more plant genomes On line discussion group (https://groups.google.com/forum/#!forum/talengi- neering ; (https://groups.google.com/forum/#!forum/crispr). (Jorge Lozano, 2014) 27

38. 40 General protocol for CRISPR

39. 41

40. What makes CRISPR system the ideal genome engineering technology 42

41. Examples of crops modified with CRISPR technology 43 CROPS DESCRIPTION REFERNCES Corn Targeted mutagenesis Liang et al. 2014 Rice Targeted mutagenesis Belhaj et al . 2013 Sorghum Targeted gene modification Jiang et al . 2013b Sweet orange Targeted genome editing Jia and Wang 2014 Tobacco Targeted mutagenesis Belhaj et al . 2013 Wheat Targeted mutagenesis Upadhyay et al . 2013, Yanpeng et al. 2014 Potato Soybean Targeted mutagenesis Gene editing Shaohui et al ., 2015 Yupeng et al., 2015 Harrison et al ., 2014

42. Application in Agriculture Can be used to create high degree of genetic variability at precise locus in the genome of the crop plants. Potential tool for multiplexed reverse and forward genetic study. Precise transgene integration at specific loci. Developing biotic and abiotic resistant traits in crop plants. Potential tool for developing virus resistant crop varieties. Can be used to eradicate unwanted species like herbicide resistant weeds, insect pest. Potential tool for improving polyploid crops like potato and wheat. 44

43. Some pitfalls of this technology Proper selection of gRNA Use dCas9 version of Cas9 protein Make sure that there is no mismatch within the seed sequences(first 12 nt adjacent to PAM) Use smaller gRNA of 17 nt instead of 20 nt Sequence the organism first you want to work with Use NHEJ inhibitor in order to boost up HDR 45 Solutions Off target indels Limited choice of PAM sequences

44. How to avoid off-target effects? Optimization of Injection conditions (less cas9/ sgRNA ) Bioinformatics : Find a sgRNA target for less off-targets “CRISPR Design” (http:// crispr.mit.edu ) 46

45. Final Conclusion Crop improvement requires the constant creation and use of new allelic variants Genetic modification, including plant breeding, has been widely used to improve crop yield and quality, as well as to increase disease resistance Progress in site specific nuclease coupled with increase crop genome sequencing and more effective transformation system offers great promise in creating non transgenic crops with predetermined trait TALENs, CRISPR/ Cas , and ZFN can be easily fashioned to bind any specific sequence of DNA (TALEs, CRISPR/ Cas ) because of the simple rules governing their interactions with nucleic acids.

46. Using these technologies ( ZFNs, TALENs, and CRISPR/Cas9 ) plant genome can be successfully modified Among the different genome editing tools after TALEN, CRISPER/Cas9 is getting more popularity owing to specticity simplicity ease in construction A step ahead from the fear of transgenic. Final Conclusion

47. Thank you