Gene editing is a type of genetic engineering that modifies an organism's DNA by deleting, inserting or replacing parts of the genome. The document discusses several molecular "scissors" or nucleases that can make targeted cuts in DNA, including meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR-Cas9 system. These nucleases are used to induce double-strand breaks that are then repaired through natural cellular mechanisms, allowing for targeted changes to the genome. The techniques have been applied successfully in several crop plants to develop traits like herbicide resistance and disease resistance.

1. Gene Editing: An Essential Tool
For Plant Breeding
Noreen Fatima
2011-ag-2109
PhD Scholar
Supervisor: Dr. Asif Saeed

2. Contents
 What is Gene Editing?
 Molecular Scissors
 Meganuclease
 ZFNs
 TALENs
 Crisper Cas9
 Outcomes

3. What is Gene Editing?
 Gene editing, or genome engineering, or genome editing, is a type of genetic
engineering in which DNA is inserted, deleted, modified or replaced in the
genome of a living organism.
 It is targeted mutagenesis (Silva et al., 2011)
 Site-Directed Nucleases (SDN):
 Zinc Finger
 TALENs (transcription activator-like effector nucleases)
 CRISPR/Cas9 systems (Clustered Regularly Interspaced Short Palindromic
Repeats - associated protein-9 nuclease (Cas9))

4. How Does It Work?
Natural repair mechanisms
4
targeted cut
molecular “scissors”
Double DNA strand break (DSB)
Specific genomic site
(e.g. gene)

5. Molecular “Scissors”
 Targeted DNA-cutting enzyme (nuclease)
1. DNA-binding domain
2. Cutting domain
 Changes: two possibilities
1. Small deletion
2. Exchange of DNA with another piece of DNA, e.g. desirable gene variant (allele)
 Meganucleases ~1980 (Marcaida et al., 2008)
 Zinc Finger Nucleases ~2005 (ZFN)
 TALENs ~2009 (Doetschman et al., 1987)
 CRISPR-Cas9 ~2012

7. 1. Meganucleases
 discovered in the late 1980s (Silva et al., 2011)
 First tool used for double strand break-induced genome manipulation
(Goubel et al., 2006: Hinz et al., 2005)
 are endonuclease enzymes, cut large DNA sequences (from 14 to 40bp).
(Paques et al., 2007)
 Occur naturally eg. I-Scal in Yeast and I- CreI in Chlamydomonas
(Redondo et al., 2008)
 Binding site and restriction site occur within same unit (Fajardo-Sanchez et
al., 2008)
 cause less toxicity in cells than methods such as Zinc finger nuclease
(ZFN), likely because of more stringent DNA sequence recognition.

8. 1. Meganuclease
 Drawback is the construction of sequence-specific enzymes
for all possible sequences is costly and time consuming
(Munoz et a., 2010)
 Difficult to manipulate DNA binding site
 Crop where it is useCrop/Plant Trait Reference
Maize Herbicide Resistance Gao et al., (2010)
Cotton Herbicide and insect
resistance
D’Halluin et al., (2013)

9. 2. Zinc Finger Nuclease
 Are hybrid restriction enzyme (Caroll et a., 2006: Smith et al., 2000)
 creating dsDNA break at specified location (Morton et al., 2006)
 Have two functional domains
 DNA binding domain: at N-terminal, chain of two finger modules
 DNA cleavage domain: at C-terminal, nuclease domain of Fok 1 (Bitinaite
et al., 1998)
 Recognize unique hexamer (6bp) sequences in DNA (Awin et al.,
2005)
 Two-finger modules stitches together to form a Zinc finger protein,
each with specificity of ≥18 bp (Beumer et al., 2008)

11. How to construct Zinc Finger Nuclease
1. Choose a DNA segment of interest and designing the coding
sequence for zinc finger protein binding to it
2. Take nonspecific cleavage domain from the FokI restriction
endonuclease (Ashworth et al., 2006)
3. These coding sequences are linked to that of the nonspecific
cleavage domain from the FokI restriction endonuclease with the
help of spacer, add nuclear localization signal (Bozas et al., 2009)
4. Clone in binary vector or expression vector (Cai et al,, 2009)
5. Test in transformation or in vitro activity

12. 2. Zinc Finger Nuclease
 Limitations
 Off target effect
 Negative impact on cell proliferation
 Construction is cumbersome and time consuming

13. Applications of ZFN
 Repairing mutations
 Insertion of gene or DNA fragment at specific site
 Repair or replace aberrant genes
 Disabling an allele
 Allele editing
 Applications in medical sector
a) Gene therapy
b) Treatment of HIV

14. 3. TALENs :Transcription Activator-like Effector
Nucleases
 First time reported in Xanthomonas oryzae (2011)
 TALENs are the restriction enzyme engineered to cut specific sequences of DNA
(Bogdanove and Voytas 2011)
 Cause double stranded DNA breaks (Christian et al., 2010)
 Consist of TALE + Endonuclease
 They are made by fusing:
 DNA-binding domain (TAL effector): have highly conserved 33-
34a.a
 DNA-cleavage domain ( the catalytic domain of RE FoK I): function
as dimer
 can be engineered to bind any desired DNA sequence to cut at specific locations
 Use for treatment of various diseases (Sun et al. 2012)

15. Molecular Structure of Effector
 TAL effectors are organized into3 sections
1. N-terminal domain: have type III section signal (Botch et al., 2009)
2. A central domain: help in DNA binding specificity
3. C-terminal domain: have NLS and acidic active domain
 A stretch of 34 a.a is for these 3 domains, is repeated at 15.5-19.5 times
 In each repeated 12 and 13th a.a is vary so called repeats variable diresidues
(RVDs)
 Amino acid identity in RVDs is responsible for DNA nucleotide recognition
and enabling design of TALENs to target DNA sequences (Doyle et al.,
2012)

16. 3. TALENS
 Each amino acid recognizes one nucleotide of the target DNA
sequence (Cermak et al., 2011)
 And have three advantages in targeted mutagenesis:
1. DNA binding specificity is higher
2. off-target effects are lower
3. construction of DNA-binding domains is easier
 Based on the maximum theoretical distance between DNA
binding and nuclease activity, TALEN approaches result in the
greatest precision (Revon et al., 2012)

17. 4. CRISPR Cas9
 Clustered regulatory interspaced short palindromic repeats (Bortesi
et al., 2015)
 Segments of prokaryotic DNA, have repetitive base sequence
 Bacteria use it for defense system
 CRISPR array is composed of series of repeats interspaced by spacer
sequence acquired from invading genomes

18. Genome Editing: CRISPR/Cas9 System
• Single guide RNA (sgRNA) bound to a
nuclease (Gao et al., 2017)
• Complex goes through DNA until
finding a match (Gomez et al., 2017)
• A conformational change activates the
nuclease
• Double stranded DNA is cleaved
(Ishizaki et al., 2016)
• DNA is repaired by the cell
http://www.clontech.com/US/Products/Genome_Editing/CRISPR_Cas9/Resources/About_CRISPR_Cas9

19. Components of CRISPR
1. PAM: Proto spacer adjacent motif that act as binding site
for Cas9 protein (2-6bp in DNA)
2. crRNA/spacer : define the genomic target of cas9
3. tracrRNA: link with crRNA and serve as binding scaffold
for Cas nuclease
4. sgRNA (crRNA + tracrRNA): small RNA to guide the nuclease
5. Cas9: endonuclease use to cut the target DNA

21. Comparison of ZFN, TALEN, CRISPR-Cas9 Technologies

22. DNA Repair Mechanism
NHEJ
 Non homologous end joining
 Produces a small insertion or deletion
(without the use of exogenous DNA)
(Liu et al., 2012: Takata et al., 1998)
 breaks ends can be ligated without a
homologous template
HEJ
 Homologous directed joining
 Can introduce a desired DNA
sequence or gene into a targeted site
(Čermák et al., 2015)
 Only used by the cell when
homologous piece of DNA present in
nucleus

23. Natural repair mechanisms
Double strand break (DSB)
repair by the cell
homologous
recombinationNon-homologous end
joining (NHEJ): imprecise
precise repairmutation
repair
template

24. Outcomes of Gene Editing in PlantsCrop Gene editor Target gene DNA
repair
type
Target trait Referenc
Maize ZFNs ZmIPK1 HR Herbicide tolerant and phytate reduced
maize
Shukla et al., 2009
Rice TALENs OsSWEET14 NHEJ Bacterial blight resistance Liu et al., et al., 2012
Wheat TALENs TaMLO NHEJ Powdery mildew resistance Wang et al., 2014
Maize TALENs ZmGL2 NHEJ Reduced epicuticular wax in leaves Char et al., 2015
Tomato TALENs ANT1 HR Purple tomatoes with high anthocyanin Čermák et al., 2015
Tomato CRISPR/Cas9 SlMLO1 NHEJ Powdery mildew resistance Nekrasov et al., 2017
Tomato CRISPR/Cas9 SlJAZ2 NHEJ Bacterial speck resistance Ortigosa et al., 2018
Tomato CRISPR/Cas9 SP5G NHEJ Earlier harvest time Soyk et al., 2017
Tomato CRISPR/Cas9 SlAGL6 NHEJ Parthenocarp Klap et al., 2017

25. Outcomes of Gene Editing in PlantsCrop Gene editor Target gene DNA repair type Target trait Referenc
Rice CRISPR/Cas9 ALS HR Herbicide resistance Sun et al., 2016
Rice CRISPR/Cas9 EPSPS NHEJ Herbicide resistance Li et al., 2016
Rice CRISPR/Cas9 ALS HR Herbicide resistance Butt et al., 2017
Soybean CRISPR/Cas9 ALS HR Herbicide resistance Li et al., 2015
Maize CRISPR/Cas9 ALS HR Herbicide resistance Savistashev et al.,
2015
Potato CRISPR/Cas9 ALS HR Herbicide resistance Butler et al., 2016
Flax CRISPR/Cas9 EPSPS HR Herbicide resistance Sauer et al., 2016
Cassava CRISPR/Cas9 EPSPS HR Herbicide resistance Hammel et al.,
2018

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