Genome editing technologies allow genetic material to be added, removed or altered at specific locations in an organism's genome. Several approaches exist, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR/Cas9, and base editors. These tools create precise breaks in DNA that can be repaired through non-homologous end joining or homology-directed repair. They enable trait discovery and crop improvement by generating plants with high yield, stress resistance, or other desired properties. While powerful, challenges remain in fully editing complex genomes and reducing off-target mutations.

1. Submitted By: Saima Barki
Submitted To: Dr. Samin Shakeel
DATE: 10 JAN, 2019

2.  GENOME EDITING (also called
gene editing) is a group of
technologies that give scientists the
ability to change an organism's
DNA. These technologies allow
genetic material to be added,
removed, or altered at particular
locations in the genome. Several
approaches to genome editing have
been developed.
 Advantages
 Potential to genomic architecture,
 Precise location
 Desired acuuracy
 Efficient Uses:
 trait discovery
 Generation of crop with high yield
 Resistance to biotic and abiotic stress
 Challenges:
 edit all the genes/genome using a
particular editing tool
 Strategy:
 use of several genome editing tools
https://www.cell.com/molecular-
plant/libraries/genome-editing

3.  Homologous recombinatin (HR)
 Zinc finger nucleases (ZFN)
 Transcription activator like effector nucleases(TALEN)
 Pentatricopeptide repeat proteins (PRP)
 CRISPER/Cas 9 system
 RNA interference (RNAi)
 Cisgenesis
 Intragenesis
 Site directed sequence editing
 Oligonucleotide directed edit genome at
single nt level
 mutagenesis
 Eg. ABEs ( adenine base editors)
Mohanta et al,
Plant Physiol. Biochem. 2017,

4.  Plant domestication- 1000 years
ago
 Convention plant breading
approach
 Feeding World
 Development Of Modern Society
 Pregenomic breeding programs
 stress tolerance
 High Yield crop varieties
 Breeding program uses
 Natural selecting
favorable combinations
 Mutation induced genetic variation
 Traditional breeding program→
mutagenesis → screening
MacDonald, et al.,
Adv. Drug Deliv. Rev. 2016,

5.  Mutagensis, intergenic crosses,
traditional breeding → non-
specific
 Transgenic breeding program
→ post genomic era
 Revolution in breeding →
molecular markers
 Whole genome sequencing
 Transcriptome sequencing
 Single nucleotide
polymorphism
 Random Amplified
Polymorphic DNA (RAPD),
 Restriction Fragment Length
Polymorphism (RFLP),
 Amplified fragment length
polymorphism (AFLP )
 Single sequence repeat (SSR)
MacDonald,etal.,.Adv.DrugDeliv.Rev.
2016

6.  Combination of genomic +
conventional breeding tools=
new door opening
 System biology+ molecular
markers=identifiation of
agronomic traits
 Synthetic biology tools=
genome editing tools
 Benefits:
 precision
 Accuaracy
 Predectibility
 Do away with messiness of
inaccuracy
 Requiremnt:
 Complete understanding of
biological processes
Wang et al., 2015

7. Gene delivery is the process of introducing
foreign genetic material, such as DNA or
RNA, into host cells.
Genetic material must reach the nucleus of
the host cell to induce gene expression.
Should remain stable within the host cell
and can either integrate into the genome
or replicate independently of it.
Requires foreign DNA to be synthesized as
part of a vector,
Deliver the transgene to that cell's genome.
 Used transposon or
reterotransposon
 ↓
T-DNA →Random insertion
Single nucleotide insertion not
possible by this mechanism for
which:
 Chemical mutagenesis
led to off target mutatation
 Target induced local lesion in
gene
Kurowska et al., 2011

8. B W YAN , et al., 2013

9.  Non-homologous end
joining (NHEJ) repairs double-
strand breaks in DNA.
 NHEJ is "non-homologous as
break ends are directly ligated
no need for a homologous
template, Natural
 More efficient
 Highly conserved
 Minor or no error rate
 Can be initiated at specific sites
 Great platform for gene
targettting
 Recombination hotspot
 Chromosome
 ↓
 Double strand breaks (by
SPO11 complex i.e. topoisomerase+MRN
complex)
 ↓
 Meiotic recombination
(during cell division)
Henikoff, et al., 2014

10. Roy .s , 2014

11.  Non-homologous end joining (NHEJ) is a
pathway that repairs double-strand breaks
in DNA. NHEJ is referred to as "non-
homologous" because the break ends are
directly ligated without the need for a
homologous template, in contrast
to homology directed repair, which
requires a homologous sequence to guide
repair.
 Different types:
 Classical
 Alternate
 Alternate endjoining mechanism (AEJM)
 Disadvantage:
 Repetative nature of plant genome so slow
NHEJ rate in plants
 HDR- Specific genome targetting
Sung, P., et al., 2006

13.  Zinc-finger nucleases (ZFNs):
 artificial restriction enzyms
 generated by fusing a zinc finger
DBD to a DNA Cleavage
Domain.
 can be engineered to target
specific desired DNA sequences
 Nucleases to target unique
sequences within
complex genomes
 Use endogenous DNA repair
machinery,
 Can be used to precisely alter
the genomes of higher
organisms. Fell, V.L ., 2015

14.  STRUCTUE:
 cis2His2- 30 aa-β β’ α
 DOMAINS:
1. DBD-2 zinc finger moduels-6 bp recongnition seq
2. DCD (DNA cleavage domain)
3. Methylation domain
4. FOK1-cleavage domain/N
5. Transcrioption activation domain(A)
6. Transcription repressor domain(R)
7. Zinc finger protein (ZFP)
 FEATURES:
 Highly specific genomic scissors
 Site specific DSB
 Permenant editing, ligating DSB
 Zn ion required for chelation
 Binding site in major groove
 aa involved in DNA binding are -1,+1,+2,+3,+4,+5,+6
 Each finger bind triplet seq
Walker, J.R., et al., 2001

15. UMass Medical School

16.  Triplet recongition DNA seq are 5’-
GNN-3’, 5’-CNN-3’, 5’-ANN-3’ and
5’-TNN-3’
 Asp at 2nd position of α-involve in
cross strand-outside triple-overlap
so recognize 4 bp instead of 3 bp
 Asp increases specificity and
binding affinity
 FOK1-Type ll restriction
endonuclease-bind to palindromic
seq and cleave 9/13 nt downstream
of binding seq i.e. 5’-GGATG-3’
 3’-CCTAC-5’
 Binding signal to endonucleases
 Then cleavage take place
 Target seq are 5’-GNNGNNGNN-
3’
 Mutation are perminant and
heritable
Knoll, A et al., 2012

18.  Transcription activator-like effector nucleases (TALEN) are:
 Restriction enzymes
 can be engineered to cut specific sequences of DNA.
 Made by fusing a tal effectors dna binding re to a dna cleavage domain
(nuclease which cuts dna strands).
 (Tales) can be engineered to bind to practically any desired dna sequence
 When combined with a nuclease, DNA can be cut at specific locations.
 Used for: gene editing
 Alternative to zfns.
 Use dsb like zfns.
 Also contain non-specific endonuclease fok1
 DBD contains 30 copies of 33-34 aa, higghly conserved except at 12th and
13th position known as RVD (repeat variable diresidue), involve in
specific nucleotide recognition.
 Each repeat recongnieze single base, so potentail for versatile engineering
to create recongnition sequenice for any dna seq.
 Fok1 as dimer
 No. Of residues between DBD and FOK1 and no. Of bases between two
separate TALEN, very important in modelling the activity and affinity.

19. TALEN construction
↓
Transfer in to plasmid vector
↓
Transform into target cell
(as mRNA so eliminating chances of genomic
integration and also help in enhancing HDR
and gene expression in rice, to make it
disease resistant.)
↓
Gene product expression
↓
Enter into nucleus
↓
Necessory editing of genome
Highest cleavage rate in TALEN as compared to
ZFN,
Knock out of Arabidopsis thaliana by TALEN
Challenge is creating TALE repeat. Yin, P.; Li, Q. et al; 2013

22.  In organelle, great array of RNA
binding proteins→regulate gene
expression →post transcriptional
 PPR →charectorized by 35 aa TRM
 Different classes depending on no. of
TRM (no. of aa)
 PLS type of DYW domain at C terminal
→editing domain-zinc binding
→catalysis editing.
 PPR-6,1 Position →determine nt to
which P
 PR bind.
 Bind to 5’ of target DNA in parallel
fashion.
 Vander wall’s forces
 6,1 →Threonine, Asparagine
→recognize Adenine
 6,1 → Asparagine, Aspartic Acid
→recognize uracil
 aa at 3 (Hydrophobic) → interaction of
PPR with tRNA
De Longevialle, A.F, et al., 2008

23. Xie, K.; Yang, et al., 2013

24.  Family of DNA seq in bacteria
 Derived from virus, that had attacked
bacteria
 Used to recognize and destroy DNA from
further attacks, so protect themselves.
 Typical bacterial immune system
 Give resistance to foreign genetic material.
 Cas 9"CRISPR-associated 9") an enzyme
uses CRISPR sequences as a guide to
recognize and cleave specific strands of
DNA complementary to the CRISPR
sequence.
 Cas9 enzymes +CRISPR sequences form
the basis of a technology known
as CRISPR/Cas9 to edit genes within
organisms
 wide variety of applications.
Xie, K.; Yang, et al., 2013

25.  COMPOSITION:
 Crisper
RNA(crRNA),
 Transactivation
RNA (tracRNA),
 Cas9 nucleases,
 Protospacer
adjacent
motif(PAT),
Feng, Z, et al.; 2014

26.  Intergrate foreign RNA into cluster, produces crRNA→40 nt
long contianing PAM (complementory).
 crRNA hyberdize with tracrRNA=guide RNA(gRNA).
 gRNA activates Cas 9 system.
 gRNA-20 nt at 5’-direct cas9nuclease to complementory target
DNA( DNA-RNA complementory bp).
 Requisite for cleavage: PAM motif( 5’-NGG-3’ or 5’-NAG-3’)
downstream of target DNA.
 Specificity by 12 nt. Seed sequence upstream of PAM, should
match between RNA and DNA.
 Cas9→DSB →NHEJ or HDR
 1 Cas9 and multiple gRNA= more than 1 site can be targetted
and alter simultaneously.
 So when 1 gRNA is inefficinet at disrupting a targetted gene or
altering more than one gene at a time.

27. ROYAL SOCIETY FOR
BIOLOGY

28. ROYAL SOCIETY FOR
BIOLOGY

29.  DISADVANTAGES:
 High frequency of off
target mutations but rare in
plants
 1.6% in rice, but no off
target in oat, thaliana and
wheat.
 8-12 nt at 3’of gRNA
determine the specificity.
 At 5’ mismatches are
tolerable as compared to 3’
 Increase GC decreases the
off target mutations.
 <30% GC content –
increases the off target
mutations.

30.  Mutate A-T bps to G-C bps.
 Use tRNA
deoxyadeninedeaminase (Tad
A), with catalytically impaired
Cas9 nucleases to mutate AT
bps to GC bps.
 In human and bacteria
 High accuracy and purity.
 More efficient in point
mutations as compared to Cas9
nucleases.
 High product purity >99.9 %
 Low rate of indels
 Doubling the linker seq leads to
efficiency of editing.
 Limited editing in case of
multiple As
Burch-Smith, et al., 2016

31. AMERICAN CHEMICAL
SOCIETY

32. Addgene.org

33.  Site-directed mutagenesis/site directed sequence editing:
 method that is used to make specific and intentional changes to
the DNA sequence of a gene and any gene product.
 used for investigating the structure and biological activity of DNA,
RNA and protein molecules, and for protein engineering.
 For example;
 Post-transcriptional modification of C to U
by deaminase Apolipoprotein B mRNA
editing
enzyme, catalytic polypeptide 1
(APOBEC1) in combination with RNA-
binding protein A1CF. RMB47
is necessary for APOBEC1 mediated editing
NEB

34. Tan, S.; Evans, R.R.; et al.,
2016

35. NEB

37.  RNA
interference (RNAi):
 biological process in
which RNA molecules
inhibit:
 gene expression or
translation,
 by neutralizing targeted
mRNA molecules.
 Other names include co-
suppression, post-
transcriptional gene
silencing (PTGS), and
quelling.
Iyer, L.M. 2013;

38. Gasparis, et al., 2017

39.  Cisgenesis :
 Cisgenesis (from "same" and
"beginning") is one term for
organisms that have been
engineered using a process:
 Is the introduction of isolated
genes with their native promoters
from crossable species.
 Genes are only transferred
between closely related organisms.[
 The term was first introduced in
2000 by Henk J. Schouten and
Henk Jochemsen
 in 2004 a PhD thesis by Jan Schaart
of Washington
university discussing
making strawberies less
susceptible to Botrytis cineres.

40.  Transfer of genes between corssable species.
 An alternative to transgenetics
 Unlike cisgenes, intergenes are hybrid genes.
 They can have genetic elemnets from different genes
and loci.
 By using different promoter or terminator regions,
expression of genes can be modified.
Yoshinaga, K, 2017;

42.  ZFNs and DSBs can potentially be used for precise genome editing in
plants, and can
have a huge impact in functional genomics studies. Very helpful in plant
trait discovery and improving commerical plants.
ZFNs and TALENs are routinely used in plant research that uses HDR-
based mutations, produce unwanted results and lead to HDR-altered
alleles and alos can potentially be used to create fusion proteins
containing domains other than
nucleases.
 TALE array repeats can be used in order to induce epigenetic
modifications in specific genomic regions to induce stable and heritable
mutations. Lack of sufficient genetic data set to
address the sequence specificities for different genome editing tools is the
biggest limitation for
target prediction.

43.  The CRISPR/Cas9 system can be very useful for
post-transcriptional control of gene expression.
 ABE-mediated genome editing will be very useful
for generating point mutations/deletions with
high accuracy and less indels.
 Chromosome engineering and the synthetic plant
genome approach can be used as a potential tool
for DSB-mediated genome editing in future. These
genetic devices can be integrated into the genetic
circuit to switch on and off to a particular trait or
pathways.

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