Targeted mutagenesis is a molecular biology technique used to intentionally induce specific changes to the DNA sequence of a gene. Zinc finger nucleases (ZFNs) are one method for targeted mutagenesis. ZFNs consist of a DNA-binding domain containing zinc finger motifs and a DNA-cleavage domain from the FokI restriction enzyme. The zinc finger domain binds to a specific DNA sequence while the FokI domain cleaves the DNA, creating a double-strand break. This double-strand break stimulates the cell's natural DNA repair processes and can result in targeted gene deletions, insertions or modifications.

1. Targeted Mutagenesis and Mutation In
Crop Improvement
CREDIT SEMINAR
ON
PRESENTED BY
Sourav Ranjan Nanda
M.Sc. Scholar
1

2. Global
warming
Recurrent
floods
Drought
Disturb
Rainfall
Abiotic stress
Biotic stress
2

3. Absolute yield
Stability of yield
Improved quality
3

4. MUTATION-Sudden heritable change in genetic material
or character of an organism
MUTANT- Individuals showing mutation
VARIANT -An individual showing an altered phenotype
due to mutation.
MUTAGEN -Factor or agents causing mutation are
known as mutagen.
Gene mutation/Point mutation- Mutation which
causes changes in base sequence
4

5. 1791 Seth Wrigh mutation first time male lamb with unusual short legs
1900 Hugo de Vries Term mutation Oenothera lamarckiana.
1910 T.H.Morgan Systematic study of mutation white eye mutant of Drosophila
1927 induced mutation by using x ray
H. J. Muller
Drosophila
1928 ,L.J. Stadler barley and maize.
induced mutation by using x ray
1939
R.A. Steinberg
Aspergillus
Induction of mutations by chemicals
1946 C. Auerbach and
J.N. Robson
Induction of mutations by chemicals in Drosophila.
5

6. 6

7. Lethal mutation DEATH 100%
Sub lethal mutation DEATH 90%
Sub vital mutation DEATH <90%
Vital mutation DEATH 0%
Super vital mutation SURVIVAL Enhance
Based on the survival of an individual.
7

8. Based on causes of mutation
• occurs naturally
• Eukaryotes>prokaryotes
Spontaneous
Mutation
• either a chemical or physical agent
• Use of induced mutation for crop
improvement program is known as
mutation breeding.
Induced
Mutation
8

9. Based on tissue of origin
• somatic cell
•Transmit to progeny
Somatic
mutation
• Gametic cells or
reproductive cells
•It is not transmitted
Germinal
Mutation
9

10. Normal/wild type
Mutant allele
Forward
mutation
Backward
mutation
Based on direction of mutation
10

11. Dominant Recessive
Wild allele
Dominant
mutation
Recessive
mutation
Phenotypic effect
Wild allele
Co-dominant mutation
Co-dominant nature
11

12. • Chromosome Mutations may Involve: –Changing the structure of a
chromosome due to loss or gain of part of a chromosome
12

13. Molecular basis of mutations
Point mutations Base substitution
Base deletion
Base addition
13

14. Base substitution:-
14

15. Base deletion
Base addition
• .
15

16. 16

17. Mutations in case of Crop Improvement
Spontaneous
mutations
Due to error during DNA replication
Due to mutagenic effect of natural
environment
occur naturally
17

18. Crop
Rice
Wheat
Groundnut
Sorghum
Variety
GEB-24
Norin
TMV-10
Co-4
18

19. Artificial induction of mutations: Mutations can be induced
artificially using
1. Physical mutagens or radiations
2. Chemical agents
Physical mutagens:-
Ionizing
radiations:
Non-particulate
Produce free
radicle
Example-X-rays and 𝛾 −
𝑟𝑎𝑦𝑠
Particulate
Ionization and
Excitation
Eg :,𝛼 − 𝑟𝑎𝑦, 𝛽 −
𝑟𝑎𝑦, 𝐹𝑎𝑠𝑡 𝑛𝑒𝑢𝑡𝑟𝑜𝑛
Non-ionizing
radiations.
Pyrimidine dimer
Example: UV
rays.
19

20. 20

21. Chemical
mutagens
Alkylating
agents
Dimethyl sulphonate (DMS), Ethyl methane sulphonate
(EMS),Nitroso methyl Urea (NMU), Nitroso ethyl Urea
(NEU), Methyl methane sulphonate (MMS).
Acridine
dyes
proflavine, acriflavine,
Acridine orange,
Deamination Nitrous acid
Base
analogues 5– bromouracil, 3-bromodeoxy
uridine, 2 -amino purine.
21

22. Mainly three types screening/selection techniques are
used in M2 and subsequent generation.
• Visual: Most effective and efficient method for identifying
mutant phenotypes.
• Mechanical/Physical: Very efficient for seed size, shape,
weight, density, etc. using appropriate sieving machinery.
• Other methods: Low alkaloid content mutants can be
selected using colorimetric tests. Chromatographic or
electrophoresis techniques may be used to select isolate
protein variants.
22

23. Handling of segregating generations
Dose of mutagen
Part of the Plant to be Treated
Choice of mutagen
Choice of material
Procedures of Mutation Breeding
23

24. Choice of material :
It should be the best variety available in crop and seed should
be pure.
Choice of mutagen :
Generally chemical mutagens are more preferred for seed
treatment and radiations for the treatment of vegetative parts.
Part of the Plant to be Treated
Seeds
Pollen grains
Vegetative propagules
Corns Bulbs
Complete plant
24

25. Dose of mutagen
• Mutagens generally induce a high frequency of chromosomal changes meiotic and mitotic
irregularities.
• Optimum mutagen dose is one, which produces maximum frequency of mutations and causes
the minimum killing.
• Close to LD50 dose is optimum.
• LD50 is the dose of mutagen that kills 50% of the treated individuals.
• EXAMPLE:- EMS – 0.3-1.5 %, for 2-6 hours
Handling of segregating generations
• Mutation breeding for oligogenic traits
• Mutation breeding for polygenic traits
25

26. Mutation breeding for oligogenic traits
I. Treated seed are space planted
II. Seed from individual plants are harvested separately
I. Individual plant progeny are grown
II. Seed from all the plants of each row containing or suspected to
contain the mutant alleles harvested separately
I. Individual plant progeny are grown
II. Superior mutant lines are harvested in bulk if they are
homogenous
I. Preliminary yield trail with superior check
II. Superior mutant lines selected
I. Replicated yield trail at several location
II. Outstanding line released as anew variety
I. Seed multiplication for distribution among farmers
26

27. Mutation breeding for Polygenic traits
I. Treated seed are space planted
II. Seed from individual plants are harvested separately
I. Individual plant progeny are grown
II. Fertility,vigrous,normal looking plant harvested separately
I. Individual plant progeny are grown
II. Superior mutant lines are harvested in bulk if they are homogenous
I. Preliminary yield trail with superior check
II. Superior mutant lines selected
I. Replicated yield trail at several location
II. Outstanding line released as anew variety
I. Seed multiplication for distribution among farmers
I. Individual plant progeny are grown
II. Superior plants selected from superior progenies showing segregation
III. Homogenous mutant progenies harvested in bulk
27

28. 28
GENETIC ENHANCEMENT OF RICE

29. 29
KT 20-74
SH 30-21
China 1957
Semi dwarf
mutant Reimei
Japan
lodging resistance
first rice varieties

30. Crop Mutant variety Released year Mutagen
Cotton MA-9 1948 Gamma rays
Wheat NP-836 1961 X-rays
Sugarcane Co-6608 1966 Gamma rays
castor Aruna 1969 Neutron
Black gram Co-04 1978 MMS
SESAMUM KALIKA 1980 EMS
LENTIL S-256 1981 Gamma rays
COWPEA V-16 AMBA 1981 DMS
MUNG BEAN TAP-7 1981 Gamma rays
CHICKPEA Pusa-408 1985 Gamma rays
30

31. means to synthesize something
31

32. 32
Random mutagenesis-
mutation can be generated , at any
region of DNA sequence, randomly
Targeted Mutagenesis
Targeted mutagenesis is a molecular
biology method that is used to make
specific and intentional mutating changes
to the DNA sequence of a gene and
any gene product

33. Targeted Mutagenesis
• Targeted mutagenesis is a molecular biology method that is used to make specific
and intentional mutating changes to the DNA sequence of a gene and any gene
product.
• Gene targeting is mostly achieved by homologous recombination or non-homologous
end joining.
• The efficiency of non-homologous end joining is better than homologous
recombination for gene targeting.
• Some strategies have been developed to enhance the homologous recombination by
overexpression of RAD54, recA and ruvA proteins which are involved in
homologous recombination.
• Nucleases induce the double-strand break at the target site which is then repaired by
homologous recombination or non-homologous end joining, depending on the type of
mutation to be introduced (insertion or deletion)
33

34. Targeted
Mutagenesis
Transcription
activator-like effector
nucleases (TALENs)
Oligonucleotide
-directed gene
targeting
CRISPR /cas 9
ZINC
FINGER
NUCLEASES
(ZFNS)
34

35. • To study changes in protein activity
Change in codon change in Amino Acid Change in
protein structure
• To introduce or remove restriction endonuclease sites or tags.
• To select the mutations that have desired property.
35

36. 36
ZINC FINGER NUCLEASES
(ZFNs)

37. 37
Amino acids are building blocks of
protein which are joint together to make
alpha helix and beta plated which are
secondary structure. The secondary
structure further rearranges themselves
rearrange to make the tertiary structure.
The tertiary structure contains different
type of domain which are interact
themselves.
A Specific motifs/domain found which
contains a zinc in the domain which
interact with all the structure present in
domain/motif.

38. 38

39. 39
WHAT IS ZFN TECHNOLOGY??
 A class of engineered DNA-binding proteins
 Facilitate targeted editing of the genome by creating double-strand breaks in DNA at user-specified
locations.
Double-strand breaks are important for site-specific mutagenesis.
Stimulate the cell's natural DNA-repair processes, namely homologous recombination and non-
homologous end joining (NHEJ).
Generate precisely targeted genomic edits, resulting in cell lines with targeted gene deletions,
integrations, or modifications.
Zinc fingers were first discovered in the African clawed toad (Xenopus laevis) in 1985.

40. 40
WHAT ARE ZINC FINGER NUCLEASES?
HIGHLY-SPECIFIC GENOMIC SCISSORS
Consists of two functional domains:
a) A DNA-binding domain
b) A DNA-cleaving domain comprised of the nuclease domain of Fok I.

41. 41

42. 42
DNA CLEAVAGE DOMAIN
• FokI, naturally found in Flavobacterium okeankoites
• It is a bacterial type II restriction endonucleases consisting of an N-terminal DNA binding domain
and a C-terminal non specific DNA cleavage domain.
• FokI functions as adimer, so a set of two ZFNs are used to bind to the target DNA
• Results in a unique combinatio of 18 nucleotides.
• After its binding to the cell DNA ,the FokI domain (with digests the DNA at specific site and
creates a double-stand break and triggered the cell’s repair mechanism.
• Break is repaired by HR or NHEJ leads to deletion or insertion mutations at the repair site.
• ZFN target sites consist of two zinc-finger binding sites separated by a 5 to 7bp spacer sequence.
• This spacer is recognised by the FokI cleavage domain

43. 43
Repair outcomes of a genetic double strand break in case of ZFN cleavage

44. ZINC FINGERS CAN BE INTRODUCED INTO AN ORGANISM IN VARIOUS WAYS
Introduce zincfingers in the form of a recombinant protein (still under
development)
The gene that codes for the zinc finger effector protein can be introduced
into the cell
The most commonly used vectors are plasmids and attenuated viruses
Agrobacterium mediated transfer
Particle bombardment method (direct DNA transfer)
Injecting ZFN mRNAs and donor DNA into embryos

45. 45
VARIOUS USES OF TECHNIQUE:
 Repairing mutations
 Insertion of a gene or DNA fragment at a specific
site
 Repair or replace aberrant genes
 Disabling an allele
 Allele editing
 Applications in the medical sector
 Gene therapy
 Treatment of HIV

46. TALENS-TRANSCRIPTION ACTIVATOR
LIKE EFFECTOR NUCLEASES

47. 47
TALES: TRANSCRIPTION ACTIVATOR LIKE EFFECTOR
In 2011, named the methods of the year
TALEs are naturally occuring proteins from the plant pathogenic bacteria
Xanthomonas.
These bacteria secrete effector proteins (transcription activator like
effectors, TALEs) to increase its susceptibility to the pathogen.
These effector are capable of DNA binding and activating the
expression of their target genes by mimicking the eukaryotic transription
factors.

48. 48
Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be
engineered to cut specific sequences of DNA.
 They are made by fusing:
( TAL effector) DNA-binding domain
 DNA cleavage domain (the catalytic domain of RE FokI).
TALENs can be engineered to bind to practically any desired DNA to cut at specific locations.

49. 49

50. 50

51. 51
APPLICATIONS:
 Application in Gene Therapy
 To efficiently modify plant genomes
 To correct the genetic errors that underlie disease
 TALEN can be used as tools to harness the immune system to fight
cancers
 TALEN mediated targeting can generate T cells that are resistant to
chemotherapeutic drugs and show anti-tumor activity

52. 52
This technique was first develop by the Eric Kmiec at
Thomas Jefferson University
What is chimeraplast?
Chimeraplasty-mediated modification of a target
sequence is accomplished using an exogenous
polynucleotide, the so-called chimeraplast
Chimeraplast may be a DNA / RNA
The original chimeraplast ranged between 68 and 88
nucleotides in length and comprised both DNA and 20
-O-methylmodified RNA residues

53. 53

54. 54

55. Crispr /cas 9 technique

56. 56
RERPEATS
SPACER

57. 57

58. 58

59. 59
Distict type of CRISPR identified so far

60. 60
• Protospacer adjacent motifs
• Crisper RNA(cr RNA)
• Transactivating crispr
RNA(tracer RNA)
Guide RNA

61. 61
CRISPR/CAS SYSTEM IN PROKARYOTES
• It is the part of the adaptive immune system of bacteria and archaea, protecting them
against invading nucleic acids such as viruses by cleaving the foreign DNA
 CRISPR (clustered regularly interspaced short palindromic repeats) loci
 Cas(CRISPR aasociated) protein that can target and cleave invading DNA in sequence specific
manner
 A crispr array is composed of a series of repeats interspersed by spacer sequences acquired from
invadig genome
 The immunity is acquired by the integration of these short fragments of the invading DNA
(spacers) between two adjacent repeats of a CRISPR locus present in host genome
 The most widely used system in the type II (CRIPSR)/Cas9 system from Streptococcus pyrogenes.

62. 62

63. A proto spacer adjacent motif (PAM) is a 2–6-base pair DNA sequence immediately following the DNA
sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
 The PAM is a component of the invading virus or plasmid, but is not found in the bacterial host genome and
hence 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 which distinguishes bacterial self from non-self DNA, thereby
preventing the CRISPR locus from being targeted and destroyed by the CRISPR-associated nuclease
WHY PAM SEQUENCE IS PREREQUISITE FOR CLEAVAGE?

64. 64
•Nuclease induced double strand breaks(DSBs)
can be repaired by non homologous end joining
(HDR) pathways.
•Imprecise NHEJ mediated repair can produce
insertion and /pr deletion mutations of variable
length at the site of the DSB
• HDR-mediated repair can introduce precise
point mutations or insertions from a single
stranded or double stranded DNA donor
template

65. 65
• Domestication of crop
• Remove undesirable traits from
cassava
• remove gladin in wheat
• Resistance to fusarium-Head
blight
Thinopyran detoxify vominotoxin

66. 66
Induced phenotypic diversity in the mutagenized populations
of faba bean using physical and chemical mutagenesis.
A study was conducted by using physical and chemical mutagen in fababeans to induced
phenotypic diversity .physical mutagen like gamma rays and chemical muatagen EMS is used.
Different treatment of physical and chemical mutagen is used on faba beans seeds of Vikrant
and PRT-12 to know the phenotypic diversity.
CASE STUDY 1-

67. 67
Mutagens used-
Gamma rays (c rays)- Dry seeds of each variety, with moisture content 12%, were irradiated with 100,
200, 300 and 400 Gy of gamma rays with a radioisotope 60CO source
Ethyl methane sulphonate (EMS)- For EMS treatments, healthy seeds of uniform size of each variety
were presoaked for 9 h in distilled water and treated with 0.01, 0.02, 0.03 and 0.04% of EMS for 6 h
with intermittent shaking at room temperature of 22 ± 1 C. The solution of EMS was prepared in the
phosphate buffer of pH 7.
Combination treatment: Gamma rays + EMS-Dry seeds of each variety were first irradiated with
gamma rays at 100, 200, 300 and 400 Gy doses and then treated with 0.01, 0.02, 0.03 and 0.04%
EMS. (i.e. 100 Gy gamma rays + 0.01% EMS, 200 Gy gamma rays + 0.02% EMS, 300 Gy gamma
rays + 0.03% EMS and 400 Gy gamma rays + 0.04% EMS).

68. 68
These morphological mutation observed in plant either
by the physical and chemical or combination of both
• Tall mutants
• Dwarf mutants
• Bushy mutant
• Broad leaves mutant
• Narrow leaves mutants
• Opposite leaves mutants
• Bilobed leaves mutant
• Trilobed leaves mutant
• Elongated leaves mutant
• Trifoliate leaves mutant
• Fused leaflets mutant
• Single flower mutant
• Four flowers mutant
• Five flowers mutant
• Pink flower mutants
• Vegetative mutants

69. 69
In this study, we have demonstrated that TALENs and the CRISPR/Cas system are able
to induce targeted mutagenesis in Zea mays with high and comparable efficiencies.
Although TALENs are effective tools for genome editing, there are some limitations
regarding the potential target sites, such as the need for T at position − (and the fact
that some TALENs fail to cause mutations. The recently developed CRISPR/Cas
system seems to provide a complementary approach to TALENs, as it only requires
the PAM (NGG) motif preceding the recognition sequence.

70. 70
In addition, the CRISPR/Cas system has great advantages in terms of easy 150 cloning and multiplex
genome editing. However, the high-frequency of off-target Transcription activator-like effector
nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-
associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species.
Here, They report, for the first time, targeted mutagenesis in Zea mays using TALENs and the 17
CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, 18 ZmIPK1A, ZmIPK,
ZmMRP4, and obtained targeting efficiencies of up to 23.1% in 19 protoplasts, and about 13.3% to
39.1% of the transgenic plants were somatic 20 mutations. Also, we constructed two gRNAs targeting
the ZmIPK gene in maize 21 protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition
the 22 CRISPR/Cas system induced targeted mutations in Zea mays protoplasts with 23 efficiencies
(13.1%) similar to those obtained with TALENs (9.1%). Our results show 24 that both TALENs and the
CRISPR/Cas system can be used for genome modification

71. 71

72. 72

73. 73