• Genome: A genome is the complete set
of genetic information in an organism. It
provides all of the information the
organism requires to function
• Genome Editing: Genome editing, or
genome engineering, or gene 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 a technique that enables us to alter
the regulation of gene expression
patterns in a pre-determined region and
facilitates novel insights into the
functional genomics of an organism
Introduction
3

Time Line
1953:
Discovery
of the
Double
Helix
By: Watson
and Crick
1958: in vitro
synthesis of
DNA
By:
Kornberg
1968: Discovery
of Restriction
Enzymes
By: Arber, Smith
and Nathans
1971: rDNA
technology
By: Paul Berg
1975: Hybridoma
Technology
By: Kohler and
Milstein
1973: First Genetic
Modified Organism
(Kanamycin resistant
bacteria)
By: Boyer and Cohen
1983: First Transgenic Plant:
Tobacco
By: Bevan, Flavell and Chilton
1985: Zinc Finger
Nuclease Technology
and Meganucleases
By: Aaron Klug
1983: PCR
Technology
By: Kary Mullis
1974: First
Transgenic
Animal (in Mouse)
By: Jaenisch and
Mintz
1982: First Genetically
Engineered Human Drug
- Synthetic Insulin
By: Dennis Kleid
(Genentech Company)
1987: First GMO
released in environment
Psuedomonas syningae
in Potato field in USA
2015: Firt commercially
released GM Animal as food
i.e. AquAdvantage Salmon
By: AquaBounty Company
2010 & 2011: Discovery of
TALENs and Technology
By: Boch and Bonas, 2010
and Voytas et al., 2011
Xanthomonas spp.
2010: The World's First
Synthetic Life Form
By: Craig Venter Inst.
Synthetic life Mycoplasma
mycoides
2012: CRISPR
Genome Engineering
Tool
By: Doudna and
Charpentier
1993: CRISPR Sequence
By: Yoshizumi Ishino (in E. coli in
1987)
Fransisco Mojica (in Haloferax
and Haloarcula in 1993)
1994: First GM food released
for commercial cultivation
“Flavr Savr” tomato by
Calgene
2003: Firt commercially released
GM Animal GloFish
By: Blake and Crockett
A Zebra Fish with Fluorescence gene
5

Genome
Editing
Genome Editing by
Site Specific
Recombinases
Site Specific
Recombination
Genome Editing by Site
Specific Nucleases
(i.e. induce DSBs)
Meganuclease-
based
Engineering
Zinc Finger
Nuclease-Based
Engineering
TALEN Genome
Editing System
CRISPR/CAS
Genome Editing
System
Genome Editing by Site
Specific Nickase
(i.e. without inducing
DSBs)
Single Base
Editing
Prime Editing
Types
6

• Site-specific recombination, also known as conservative site-specific recombination, is a type
of genetic recombination in which DNA strand exchange takes place between segments
possessing at least a certain degree of sequence homology
• Enzymes known as site-specific recombinases (SSRs) perform rearrangements of DNA
segments by recognizing and binding to short, specific DNA sequences (sites), at which they
cleave the DNA backbone, exchange the two DNA helices involved, and rejoin the DNA
strands
• Site-specific recombination systems mediate certain types of genetic exchange in prokaryotes
for processes that are essential for growth and adaptation mechanisms such as control of gene
expression
• Many different genome modification strategies rely on SSRs i.e. among these recombinase-
mediated cassette exchange (RMCE), an advanced approach for the targeted introduction of
transcription units into predetermined genomic loci
Introduction
8

• Phage Integration: The integration of the phage DNA into the host genome requires that
the host and the circularized phage genomes each have an attachment site, attB (B for
Bacteria) and attP (P for Phage), respectively
• Mobile Genetic Elements: Site-specific recombination is involved in the maintenance
and mobilization of bacterial mobile genetic elements, such as plasmids, transposons,
integrons and phages
• Site-specific recombinases are also required by bacteria for the stable maintenance of
chromosomes, ensuring that each of the daughter cells receives one copy of the
chromosome
Need Biological Functions of Conservative
Site-Specific Recombination
9

SSR
Originally the system that integrates and excises the bacteriophage λ genome from
a specific site in its E. coli host genome was done by Site Specific Recombinases
Minimum catalytic requirement is a recombinase protein that recognizes and
acts on two recombination sites, which can be identical or of different sequences
Two families of recombinases have distinct mechanisms for catalysing and
regulating recombination
The recombinase binds to the recombination substrates at the specific sites
and brings them together to form a synapse within which DNA cleavage
occurs
Strand exchange then repositions the cleaved DNA ends into a recombinant
configuration to facilitate rejoining of the DNA backbone to form the products
10

SSR
The process does not require the input of any cofactors such as ATP and there is
no synthesis or degradation of DNA thus called Conservative SSR
Another distinction from HR is that site-specific recombination does not depend on
long stretches of DNA identity or similarity in the two sites.
Fig. (A) SSRs promote cleavage of DNA strands at two sites in the DNA rearrange (‘swap’) the cleaved ends,
and rejoin them in the new arrangement. (B) A typical recombination site. A crossover site (usually 30–40 bp)
with inverted repeat symmetry (indicated by the two arrows) binds an SSR dimer and contains the bonds broken
and rejoined by the SSR at its centre. The crossover site may have adjacent accessory sites which bind more
subunits of the SSR and/or regulatory proteins. (C) Outcomes of site-specific recombination. 11

• Tyrosine Recombinases: The tyrosine recombinases include phage, plasmid and
integron integrases, resolvases, invertases, and transposases. The active site of tyrosine
recombinases contains four strictly conserved amino acid residues required for catalysis:
an R-H-R catalytic triad and the tyrosine nucleophile
• Recombination involves a two-stage sequential process in which one pair of strands is
exchanged before the second pair via Holliday junction intermediate
Recombinases
Fig. |Recombination mechanism of the tyrosine recombinases: A tetramer of Cre bound to the four binding
elements brings the loxP sites together in synapse (i). the active-site tyrosines initiate exchange of two of the
four DNA strands to form a Holliday Junction intermediate (ii). Isomerization in DNA conformation then
activates the previously inactive Cre subunits which then initiate exchange of the remaining strands (iii).
Tetramer of Cre
Two loxP
sites
12

• Serine Recombinases: The serine recombinases include phage integrases,
resolvases, invertases and transposases. It consists of conserved nucleophilic
amino acid serine residue which is used to attack the DNA and which becomes
covalently linked to it during strand exchange
Recombinases
Fig. | Recombination mechanism of the serine recombinases. (A) The reaction mechanism at the
recombination sites for the resolvase/invertases and for serine integrases bringing the DNA sites together in a
synapse. (i). All four subunits are simultaneously activated to cleave the DNA, forming in each subunit a
phosphoserine link to a recessed 50-end and leaving a free 2-bp 30-overhang (ii). Subunit rotation exchanges
two DNA half sites, repositioning the half sites into a recombinant configuration. Ligation of the DNA
backbone generates the recombinant molecules (iii).
Serine recombinases
Recombination sites
2-bp crossover sequence
13

Recombinases
Fig. | Diagram of the recombinase super family. The two major families are divided based on the active
amino acid of the catalytic domain, either a tyrosine or a serine..
14

• Serine and Tyrosine bacteriophage integrases, and
sometimes other SSRs, are widely used to introduce
transgenic DNA into bacterial genomes by recombination
between a site on a transgenic plasmid and a genomic site
• The integrating sequence may contain a selectable marker
gene along with other genes of interest, for example
enzymes involved in a biosynthetic pathway
• Some strategies involve a second round of site-specific
recombination after plasmid integration, which deletes the
plasmid backbone along with any marker genes used for
selection of successful integrants, and leaves behind just the
transgene(s), giving a ‘markerless’ product strain
• SSRs have also been used as experimental tools to probe
fundamental features of DNA biochemistry in vivo (for
example the structures of compacted bacterial genomic
DNA) and in vitro (for example DNA supercoiling and
topology)
Application Microbiology:
Integrating plasmids
15

Application
• The organism being used or studied must first be genetically modified (for
example using HR or recombineering) to introduce constructs containing
one or more recombination sites for the SSR to be used, as no such sites are
likely to be present in the natural genomic sequence
• Suitable construct once integrated, can then be used efficiently and
repeatedly. In fact, in many laboratory organisms, it has become routine to
include sites for commonly used SSRs (usually Cre, FLP or φC31
integrase) along with integrated transgenes to facilitate future
manipulations, so that large collections of site-containing strains are now
available
• The design of the constructs that are introduced in this way can be very
elaborate, with several recombination sites for a single SSR, or sites for
multiple SSRs
• The simplest scenario is to introduce a ‘cassette’ comprising a transgene of
interest flanked by directly repeated recombination sites
• This gene can then be ‘knocked out’ (deleted from the genomic DNA) upon
expression of the SSR, which may be regulated by an inducible promoter
Conditional Knockouts
16

• Unique feature of recombinase-mediated excision is its ability to resolve complex
insertion sites containing multiple transgenes down to single copy structures
• This strategy requires the presence of at least one recognition site within the transgene
structure, although two sites with inverted orientation flanking the entire T-DNA appear
to be the optimal configuration
• Tandem arrays will be excised due to the presence of the multiple recognition sites in
direct orientation
• Single copy transgene structures are generally the most sought-after due to their
consistent expression pattern, stability within the genome, heritability, low occurrence of
silencing, and simplicity of structural characterization
• This procedure has the advantage of lowering the total number of transgenic plants
required in order to find a properly expressing single copy line that is heritable.
Application Resolution of Transgene
Concatomers
17

Application
Fig | Schematic representation of a recombinase-mediated resolution event. This technique uses the
recombinases’ capacity to excise DNA from between any two directly oriented recognition sites. (a) The
initial construct used for transformation. b Complex transgene integration. c Transgene resolution to a single
recognition site in the genome and non-replicating circular fragment. The recombinase can be provided in cis
or in trans position 18

• An alternative strategy is to exchange a transgene (flanked by
recombination sites) for a different gene or other segment of DNA
• RMCE using a plasmid cassette donor can be regarded as a two-step
process: the first recombination reaction integrates the entire plasmid
containing the incoming cassette, and the second reaction deletes the
original genomic cassette and the plasmid backbone, leaving behind
the new cassette
• To achieve the desired result, the two sites flanking either the
genome-resident cassette or the incoming cassette should not
recombine with each other, the new cassette should be integrated in
the correct orientation, and only a single cassette should be integrated
• A strategy that can be used to favour the desired outcome is to flank
the cassettes with different sites at the left and right ends, each of
which is recognized by a different SSR
• Alternatively, the sequences of the sites flanking the cassette can be
altered so that their central overlap sequences are non-identical;
recombination between them is then blocked
Application Recombinase-Mediated
Cassette Exchange (RMCE)
19

Application
Fig. | General RMCE schematic for a single bidirectional recombinase. The pre-existing ‘TAG’ contains a
selectable marker (Marker) and is flanked by inverted RRS recognition sites. The incoming plasmid DNA
contains a gene of interest (GOI) also flanked by inverted RRS recognition sites and is termed the exchange
‘EXCH’ cassette. To proceed forward, the second set of recognition sites are then used for site-specific
excision of the intervening DNA. This results in the switch the GOI of the ‘EXCH’ cassette for the (Marker)
of the ‘TAG’ DNA. 20

Application
Fig. | The schematic representation of gene stacking via recombinase technology. a The original ‘TAG’ DNA
contains a previously targeted gene of interest 1 (GOI1), an IRS recognition site and an RRS recognition site
b For effective gene stacking an incoming vector can be introduced into the ‘TAG’ line. c Recombination
between attB and one of the two attP sites integrates GOI2 construct into the GOI1 locus. d Induction of Cre
will remove both itself and the marker gene. The remaining genome located attP is available for future
targeted integration at the ‘TAG’ locus. f Analogous to the previous steps, the attP can be used to add a third
gene, GOI3 and bring in a new attB site for yet another round of integration.
Gene Stacking
50 %
21

Advanced in vivo applications:
‘SCRaMbLE’ method for creation of genetic diversity in yeast strains, by Cre-mediated recombination
between large numbers of loxP sites introduced into synthetic chromosomes; and methods for comprehensive
engineering of bacterial genomes and ‘Brainbow’ method for labelling individual neurons in distinct colours
with combinations of fluorescent proteins
Plasmid construction and ‘gene assembly’:
A series of E. coli plasmids contain cassettes flanked by sites for λ integrase which can be replaced by a
user-specified gene (or other DNA segment) in an in vitro reaction
The replacement gene cassette, which can be generated by PCR, is first cloned into an ‘entry vector’, and can
then be exchanged into a suite of ‘destination vectors’ that support expression in various organisms, or with
different types of regulation. The system can also be used for the ordered assembly of several genes at a
single locus
Assemble DNA segments into larger arrays. SSRs of the serine integrase family have been shown to be very
useful tools for this type of operation, because the reactions between attP and attB sites are efficient and
irreversible in the absence of the RDF, and the integrases have robust activity on PCR- or oligo-nucleotide
derived substrates
Application
22

Genetic circuits, logic gates and biocomputing:
Synthetic biologists aim to create ‘genetic circuits’ that alter gene expression in a logical way upon one or
more input stimuli (such as chemical inducer molecules, heat or light). These devices can be likened to the
components and logic gates of electronic circuits
SSRs are potentially very useful for this sort of application, as they can efficiently bring about a permanent
change in the cell’s DNA sequence
Such concepts are very much still in their infancy, but one can readily imagine how logic gates such as those
already developed, and memory systems based on invertible DNA segments that can act as binary digits,
could be combined into a computing system if the specificities and efficiencies of the SSRs themselves were
sufficiently high
Nanotechnology:
SSRs have obvious potential in the field of nanotechnology, although there is limited published work to date.
For example, they could be used to implement molecular-level switches, or to attach specific DNA fragments
(perhaps with chemical modifications such as fluorophores or attached enzymes) to specific locations in
patterned 2D arrays of synthetic double stranded oligonucleotides, or to bring about site-specific
modifications of 3D structures created by the emerging ‘DNA origami’ technology.
Application
23

Off-target activity:
SSRs have very high specificity for their target sites. Nevertheless, many instances are known of SSRs
promoting low levels of ‘off-target’ recombination at ‘pseudosites’ that bear some sequence resemblance to
their natural target site(s). A notable and well-studied example is φC31 integrase, for which a number of
pseudosites (∼100) in the human genome have been identified
It is likely that some SSRs are more strictly sequence-specific than others, and protein engineering might
provide improvements.
Toxicity:
Low-level but genome-wide off-target recombination could lead to multifarious genetic problems such as
mutations, insertions/deletions and failure of chromosomes to segregate properly.
Toxicity might also be a consequence of persistence of recombination intermediates with strand breaks and
covalent SSR–DNA linkages, leading to DNA damage.
Alleviation of toxicity problems could involve judicious choice of the SSR to be used in a particular
organism or cell type, careful adjustment of SSR expression so that it is just sufficient to promote the desired
reaction, transient SSR expression, use of purified SSR protein; addition of targeting signals to the SSR for
correct (e.g. nuclear) localization, or modification of the SSR to reduce its lifetime in the cell.
Disadvantages
24

Disadvantages
Inefficiency:
Recombination may be slow and/or incomplete in some cases. These problems could be due to intrinsic
properties of the SSR, or incompatibility with the experimental system it is being used in.
For example wild-type FLP is unstable at higher temperatures (such as 37◦C), and tends to perform better in
lower-temperature organisms such as fruit flies.
The extent of recombination in eukaryotic cells is often lower than might be expected, perhaps due at least in
part to the condensed architecture of chromatinized DNA, and there may be more specific problems.
For example serine integrases can fail to complete the recombination reaction, leaving behind damaged non-
functional sites.
Substantial improvements in some of these factors might be achieved by optimization of the SSR type and
expression, including random or directed mutagenesis, codon optimization, and addition of nuclear
localization signals and/or degradation tags.
25

• One much-used ingenious approach is to fuse an SSR (for example Cre or FLP) to a steroid ligand-binding
domain, which sequesters the fusion protein in insoluble cytoplasmic aggregates until exposure to the
steroid ligand solubilizes the protein and allows it to enter the nucleus
• Other approaches include the creation of a split version of Cre (the N-terminal and C-terminal parts are
expressed separately, and can interact to reconstitute a functional recombinase), and a version of Cre that
is activated by light due to incorporation of a photosensitive synthetic amino acid analogue at the active
site
• Reconfigure the DNA recognition functions of an SSR so that it recombines at a sequence of our choice.
Most enticingly, we could then promote recombination at specific sequences in the natural genome of our
organism of interest, or even recombination between two different genomic sequences, opening the way to
systematic SSR-mediated genetic modifications for biotechnology or therapeutic purposes
• First, SSRs including Cre and FLP have been subjected to directed evolution, selecting for increased
activity on pseudo-sites having some resemblance to the natural recombination site
• An alternative strategy is to reprogramme specificity by attaching the catalytically functional parts of an
SSR to a heteroogous DNA-binding domain that targets a new sequence
Designer Recombinases
26

• A set of hybrid ZFRs comprising the catalytic (N-terminal) domain of a mutant version
of Tn3 resolvase, fused to the zinc-finger DNA-binding domain of the mouse
transcription factor Zif268, were shown to have robust activity in vivo and in vitro on ‘Z
sites’ consisting of a central sequence targeted by the resolvase
• Recently, another type of synthetic recombinase has been created by linking a serine
recombinase catalytic domain to a TALE DNA-binding domain, whose DNA sequence
specificity can be reconfigured particularly easily
Designer Recombinases
Fig. |Chimaeric serine recombinases
The figure illustrates a ZFR dimer binding to its target ‘Z-
site’. Each subunit comprises a serine recombinase domain
(SR) linked to a zinc finger domain (ZF).
27

Case Study I
Pathak and Srivastava, 2020
Arkansas, USA
28

Multigene vector, pNS64
developed with standard
restriction digestion and
ligation method
pUC with promoterless
neomycin
phosphotransferase II
(NPT II) gene between
loxP and lox75 recognition
sites
Two gene cassettes:
35S:GFP and 35S:GUS
were already available that
contained CaMV 35S
promoter driving GFP or
GUS gene
RD29a:AtDREB1a cassette
dehydration responsive element
from Arabidopsis thaliana B1A
(AtDREB1A) and Arabidopsis
cold inducible RD29a promoter
HSP17.5E:pporRFP cassette,
promoter fragment of
Gmhsp17.5E from soybean
DNA and ppor- RFP from
pANIC6A that contains coral
Porites porites RFP
Rice line T5 (Taipei 309)
that contains a Cre-lox
target site was used for
site-specific integration
lines
Southern blot analysis and
Gene xpression studies for
GFP, RFP, GUS, and
AtDREB1A
Protein analysis : NPT II
enzyme linked immunosorbent
assay (ELISA), GFP
fluorescence and GUS staining
was performed in the young
tissue
Confocal imaging :
pporRFP detection was
performed using confocal
imaging
Methodology
29

Fig. | Molecular approach for site-specific integration (SSI) of a multigene stacking. (a) T5 locus in rice cv.
Taipei-309 consisting of a single-copy of T-DNA encoding Cre activity and the target lox76 site (b) Donor
vector, pNS64, NPT II gene and four expression units (GFP, GUS, AtDREB1A, and pporRFP) between loxP
and lox75. The loxP x lox75 recombination circularize the gene construct, which subsequently integrates into
T5 locus to generate the site-specific integration (SSI) structure. (c) Structure of the predicted site-specific
integration (SSI) locus that expresses a stack of four genes (NPT II, GFP, GUS, AtDREB1A, and pporRFP).
Vector and SSI
EcoRI Digestion and Southern Blot
30

Introduction
Fig. | Transcript abundance analysis of constitutively expressed genes by real time quantitative PCR (RT-
qPCR). Relative expression of NPT II, GFP, and GUS genes in the T0 plants (a–c) and the T1 progeny
seedlings (d–e) of site-specific integration (SSI) lines in comparison to the T5 negative control. The SSI line
numbers are given on x-axis. The values are the average of two biological replicates and two technical
replicates of each. Standard errors are indicated as the error bars 31

Fig. | Protein abundance analysis
of constitutively-expressed
genes in the site specific
integration (SSI) lines.
(a–b) Quantitative GFP
fluorescence and GUS activity in
T0 plants.
(c) comparative histochemical
GUS staining of leaf cuttings
from SSI lines #10 and #12.
(d–f) GFP fluorescence, GUS
activity, and NPT II ELISA in T1
progeny seedlings. Statistical
differences, shown by the
alphabets, were determined by
student t test at p = .05. Standard
errors are indicated as the error
bars
Results
32

Results
Fig. | Protein abundance analysis of
constitutively-expressed genes in the
site specific integration (SSI) lines.
(g–h) quantitative GFP fluorescence
and GUS activity in T2 progeny
seedlings. (i) average GFP and GUS
activities in monoallelic and biallelic
T0 SSI lines (described in Table S1).
The SSI line numbers are given on x-
axis. Statistical differences, shown by
the alphabets, were determined by
student t test at p = .05. Standard
errors are indicated as the error bars
33

Fig. | Expression analysis of the inducible genes by real time quantitative PCR (RT-qPCR) in the site-specific
integration (SSI) lines relative to T5 negative control. (a–c) AtDREB1a expression analysis at room
temperature (white bars) or upon cold-induction (20 hr in ice pack, magenta bars) in T0, T1, and T2 plants.
(d–f) pporRFP expression at room temperature (white bars) or upon heat-induction (42°C for 3 hr; red bars)
in T0, T1, and T2 plants. The SSI line numbers are given on x-axis. The values are the average of two
biological replicates with standard error indicated as the error bars 34

Fig. | Confocal imaging of GFP (top) and pporRFP (bottom) in the roots and leaves of the 10 days old T1
seedlings of site specific integration lines #9, #10, and #12. All images were taken 72 hr post heat-shock
treatment at 20x magnification. T5: Target line (negative control); HS, heat-shock; RT: room temperature.
Scale bar in leaves: 100 μm and in roots: 50 μm 35

• Both Agrobacterium and gene gun are competent at multigene transfers; however, complexity of
DNA integration into the plant cell leads to multiple random outcomes consisting of complex
integrations into unique genomic sites and low rate of co-expression from multigene assemblies
poses a major bottleneck
• Recombinase-mediated site-specific integration overcomes the complexity of DNA integration and
simplifies the structure, which, in turn, removes gene silencing triggers and creates a favourable
environment for stable gene expression
• Efficiency of co-transformation with this method is exceptionally higher than generally found in
conventional methods as 19 out of 30 transformation events were successful
• All tested lines expressed each of the five genes, affording stable expression at 100% rate for the
multigene assembly
• The strong constitutive genes showed abundant expression levels and the inducible genes
functioned according to their promoter specificity
• Notably, the two inducible genes showed no apparent interference of the neighbouring genes.
Additionally, the multigene cassette was faithfully transmitted to the progeny as shown by gene
expression analysis in T1 and T2 progeny
Conclusion
36

• CRISPR - Clustered Regularly Interspaced Short Palindromic Repeats
• It is a family of DNA sequences found in the genomes of prokaryotic
organisms such as bacteria and archaea
• These sequences are derived from DNA fragments of bacteriophages that had
previously infected the prokaryote
• They are used to detect and destroy DNA from similar bacteriophages during
subsequent infections
• Hence these sequences play a key role in the antiviral (i.e. anti-phage) defense
system of prokaryotes and provide a form of acquired immunity
• CRISPR are found in approximately 50% of sequenced bacterial genomes and
nearly 90% of sequenced archaea.
CRISPR
38

• Cas9 (CRISPR associated protein 9) is a 160 kilodalton protein which plays a vital role in the
immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily
utilized in genetic engineering applications
• Cas9 is a dual RNA-guided DNA endonuclease enzyme associated with the CRISPR adaptive
immune system in Streptococcus pyogenes
• Cas9 performs this interrogation by unwinding foreign DNA and checking for sites
complementary to the 20 basepair spacer region of the guide RNA
• If the DNA substrate is complementary to the guide RNA, Cas9 cleaves the invading DNA. In
this sense, the CRISPR-Cas9 mechanism has a number of parallels with the RNA interference
(RNAi) mechanism in eukaryotes
• The Cas9 protein has been heavily utilized as a genome engineering tool to induce site-directed
double-strand breaks in DNA
• These breaks can lead to gene inactivation or the introduction of heterologous genes through
non-homologous end joining and homologous recombination respectively
Cas9
39

• CRISPR-Cas9 genome editing is carried out with a Type II
CRISPR system
Gene Editing Major Components
• Contains the guide RNA that locates the correct segment of host DNA along
with a region that binds to tracrRNA (generally in a hairpin loop form),
forming an active complex.
crRNA
• Binds to crRNA and forms an active complex
tracrRNA
• Single-guide RNAs are a combined RNA consisting of a tracrRNA and at
least one crRNA.
sgRNA
• An enzyme whose active form is able to modify DNA. Many variants exist
with different functions (i.e. single-strand nicking, double-strand breaking,
DNA binding) due to each enzyme's DNA site recognition function
Cas9
• DNA molecule used as a template in the host cell's DNA repair process,
allowing insertion of a specific DNA sequence into the host segment broken
by Cas9
Repair
template 41

Structure sgRNA/Cas9 Expression Cassettes
• A standard sgRNA contains 20-nt target
recognition sequence and plays a guiding role
for the Cas9/ sgRNA nuclease complex
• U3 or U6 small nuclear RNA gene promoters,
which recruit RNA polymerase III
• Eukaryotes, the Cas9 protein requires
attachment of nuclear localization signals for
transport of Cas9 into the nucleus
• Palindromic repeat at the end of sgRNA
according to CRISPR system
• extra small RNA that is complementary to the
repeat sequence, known as a trans-activating
crRNA (tracrRNA)
42

Structure Protospacer Adjacent Motif
• Protospacer 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
• 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
• Guide RNA (gRNA) is synthesized to perform the function of the tracrRNA–crRNA
complex in recognizing gene sequences having a PAM sequence at the 3'-end, thereby
"guiding" the nuclease to a specific sequence which the nuclease is capable of cutting
• Canonical PAM is the sequence 5'-NGG-3', where "N" is any nucleobase followed by two
guanine ("G") nucleobases
• Guide RNAs can transport Cas9 to any locus in the genome for gene editing, but no editing
can occur at any site other than one at which Cas9 recognizes PAM
43

1. Individual Expression Cassettes for Each gRNA
• Most common approach for the production of multiple
gRNAs is to express each individual gRNA by its own
promoter and terminator
• Annealed oligonucleotides are inserted into a plasmid with a
full expression cassette first
• It then encodes for distinct gRNA spacer sequences and carry
an adapter at the ends to facilitate cloning into the vector by
type-II restriction enzyme site digestions
• Afterward, the Golden Gate reaction joins the individual
cassettes to an array of multiple cassettes that can be
transferred to an expression vector
• Chief Limitation: Upto 5 RNA can be inserted due to
inefficient cloning
Multiplex CRISPR STRATEGIES
44

2. Csy4-Based Excision of gRNAs
• Csy4 is a Pseudomonas aeruginosa endoribonuclease
needed for crRNA biogenesis of the CRISPR
• Multiple gRNAs are fused together and divided by a
Csy4 excision site at their 3’end
• When co-expressing Csy4 with an artificial gRNA
array, mature gRNAs are produced that can target
multiple sites
• This method could easily be adapted for expression in
plants by providing the right Pol III promoter
• Chief Limitation: Requirement of expressing the
foreign Csy4 endoribonuclease in addition to gRNA
and Cas9
45

3. Arrays to Express Multiple crRNAs for Cpf1
• Cpf1 is a member of the class II CRISPR system that only
needs one enzyme for activity isolated from Francisella
novicida and Lachnospiraceae bacterium
• However CRISPR/Cpf1 system does not need trans-acting
crRNAs (tracrRNAs)
• Cutting specificity of this system is determined by crRNA
arrays that are processed by Cpf1 into mature single
crRNAs
• Thus Cpf1 itself can be used for crRNA processing and
gene editing and no additional nuclease needs to be
expressed
• Advantage:
1. FnCpf1 recognize a 5’-TTN-3’ and LbCpf1 a 5’-TTTN-3’
protospacer adjacent motif (PAM) in contrast to the 5’-
NGG-3’ PAM of the widely used Streptococcus pyogenes
Cas9 (SpCas9)
2. Cpf1 produces a staggered cut with a 4–5 nt overhang
46

4. Self-Cleaving Ribozyme Flanked gRNAs
• This system takes advantage of the nuclease activity of
modified ribozymes, which are RNA molecules that
catalyze reactions similar to enzymes
• Gao and Zhao (2014) designed an artificial gene called a
Ribozyme-gRNA-Ribozyme gene (RGR)
• In an RGR, the gRNA is flanked by a Hammerhead-type
(HH) ribozyme on its 5’-end and hepatitis delta virus
(HDV) ribozyme on its 3’-end
• This system could be easily modified into a multiplexed
system by tandemly joining several RGR cassettes that
could be expressed by only one promoter and terminator
at the beginning and end, respectively
• This method could easily be adapted for expression in
plants by providing the right Pol III promoter
47

5. tRNA-Dependent Expression and Processing of gRNAs by
Endogenous RNases
• Xie et al. developed an artificial gene construct that expresses
multiple gRNAs by fusing a tRNA to a gRNA and tandemly arraying
these in a polycistronic expression cassette
• The tandem arrays are expressed by a single Pol III promoter and its
terminator, and the endogenous RNaseZ and RNaseP recognize and
cut out the tRNAs, thereby releasing mature gRNAs with different
spacer sequences
• Every organism possesses Rnases that process pre-tRNAs into mature
tRNAs. Their endogenous Rnases also recognize the cloverleaf
structure of the tRNAs in the PTGs, excise them, and release multiple
individual gRNAs
• Since the tRNA processing machinery is well conserved, other
researcher have reported the effectiveness of PTGs in various plants
such as wheat and maize as well as non-plant organisms like
Drosophila, yeast, and human cells
• a gRNA driven by Pol III promoter together with a tRNA was shown
to have an increase of expression by 3–30-fold and high expression of
gRNAs also translates into higher editing efficiencies
48

6. Co-transfection of Cas9 Protein Loaded With Different gRNAs
• Purified Cas9 endonuclease is incubated with in vitro transcribed
gRNA to form preassembled Cas9-gRNA ribonucleoproteins called
RNA-guided endonuclease ribonucleoproteins (RGEN, RNPs)
• These RGEN RNPs have several advantages because they do not
require a plasmid vector or other forms of DNA for expression and
are quickly degraded after delivery into cells and editing the
genome
• This feature is especially valuable for crop trait improvement
because genome-editing RNPs will produce a mutation without the
chance of foreign DNA integration
• The Agrobacterium mediated transformation leads to transfer DNA
(T-DNA) integration from which Cas9 and gRNA are expressed
• Particle bombardment of expression vectors also leads to random
integration of the plasmid or DNA fragments into the genome.
While these foreign
• DNA integrations could be removed by backcrossing or selfing,
this is not feasible if generation times are long or if the crop is
propagated vegetatively
49

CRISPR Action
Fig. | Overview of the transfection and DNA cleaving by CRISPR-Cas9 (crRNA
and tracrRNA are often joined as a single strand of RNA when designing a plasmid)
50

Fig. | Following formation of a double stranded break (DSB), endogenous DNA repair can occur
by (A) non-homologous end joining (NHEJ) resulting in random indels, or by (B) homology-
directed repair (HDR) which uses a template DNA strand for precise repair.
DNA Repair
51

Rice MAP kinase (MKP) genes.
MPKs are important signal transducers
in plants for biotic and abiotic stresses.
Constructs were created to target
combinations of four rice MPKs with
PTGs to create a library of single,
double and quadruple mutants
Expression of two gRNAs that
target regions on the same
chromosome or gene enable the
deletion of chromosomal
fragments. Recent study deleted a
regulatory element in an intron
from the Arabidopsis flower
development gene AGAMOUS
(AG).
Li et al. replaced the second
exon of the rice ESPS gene
with a version that contains
two amino acid substitutions
(T102I and P106S) that leads
to a herbicide resistance
phenotype
Gene activation by dCas9 without
nuclease activity is fused to an
activation domain such as VP64.
Gene repression by dCas9 alone or
dCas9 coupled to repressors.
Different dCas9-based transcription
factors were tested using
agroinfiltration of N. benthamiana
leaves. They showed successful
activation and suppression of the
endogenous PDS gene
Multiplex genome editing can alleviate
some of the off-target concerns by
transforming the CRISPR/Cas9 system.
This can be achieved by fusing dCas9
with a dimerization dependent FokI
domain. Cleavage of the target site is
only possible when both FokI domains
are in close proximity to each other and
modifications need simultaneous
expression of two gRNAs to achieve
efficient genome editing
dCas9 is coupled to other
domains or proteins to enable
targeted enzyme activity to
change DNA methylation or
histone modifications. DNA
methyltransferase 3a coupled to
dCas9 was shown to induce
methylation in targeted promoter
regions resulting in gene silencing
52

Case Study II
Bahariah et al. (2021)
Bahariah et al. (2021)
53

Methodology
Design of sgRNA
In vitro validation of
sgRNA
Construction of multiplex
sgRNA CRISPR/Cas9
vector
Plant materials
Mature seeds of rice (O. sativa
L.) cultivar Nipponbare
Protoplast isolation and PEG-
mediated protoplast
transfection
Genomic DNA extraction
and mutation validation in
transfected protoplasts
Biolistic transformation of
rice calli
Genomic DNA extraction and
mutation detection in
bombarded calli
Results
54

Fig. | Fourteen-day-old rice seedlings used for protoplasts isolation (A). Stem and sheath of the seedlings
were cut into approximately 0.5-mm strips (B). The strips were treated with 0.6 M mannitol (C) followed by
enzymatic digestion. Protoplast image under the microscope (D). Bar in (D) is 100 μm (E) Schematic of
OsFAD2-1 gene loci.
A
B
C
D
E
55

PCR products
A B C
Fig. | (A) PCR product from amplification of sgRNAs template at ~ 130 bp (lane 1:
sgRNA1, lane 2: sgRNA2) for in vitro transcription
(B) PCR amplification of rice target DNA region (OsFAD2T1T2) at ~ 917 bp, and
(C) Analysis of cleavage products for sgRNA1 at 312 bp and 582 bp (lane 1) and
sgRNA2 at 614 bp and 280 bp (lane 2). Band size at ~ 130 bp in (F) at lanes 1 and
2 are the residue of sgRNA template. Red arrows indicate the band size at the
expected molecular weight 56

Fig. | Strategy for the construction of
pYLCRISPR/Cas9 targeting OsFAD2-1 in rice.
(A) Schematic illustration of target sites for two sgRNA
expression cassettes driven by U6 promoters from rice;
OsU6a promoter for OsFAD2-T1 and OsU6b promoter
for OsFAD2-T2, the 20-nt sequences in green square
boxes and orange square boxes indicate the PAM motif
of each sgRNA, the BsaI restriction enzyme sites for
the entry of multiple sgRNA expression cassettes, SP-
L2 and SP-R are sequencing vector-specific primers,
GA-L and GA-R are GA site-specific primers for the
Gibson assembly of sgRNA expression cassettes
(B) pYLCRISPR/Cas9PubiH basic vector has two BsaI
sites for insertion of two sgRNA expression cassettes
(LB: left border of T-DNA, T35S: cauliflower mosaic
virus 35S terminator, HPT: gene for hygromycin
phosphotransferase, 2xP35S: Double cauliflower
mosaic virus 35S promoter, Pubi: maize ubiquitin Ubi1
promoter, NLS: Nuclear localization sequence. Cas9p:
CRISPR associated protein 9, Nos: nopaline synthase
gene terminator, RB: right border of T-DNA, pVS1
replicon: pVS1 for replication, pBR322: pBR322
vector, KanR: Kanamycin resistance gene)
57

Fig. | PCR-amplified DNA from protoplasts
transfected with pYLCRISPRCas9Pubi-
H:OsFAD2.
(A) DNA fragments of 502 bp and 200 bp
were amplified using FAD2-F and FAD2-R
primers
(B) DNA fragments of 776 bp and 474 bp
were amplified from second set PCR using
FAD2-F2 and FAD2-R primers.
Fig. | PCR products at 474 bp were directly sequenced and analysed using SNAPGENE revealed mutations consisting of deletions in the DNA
region between sgRNA1and sgRNA2
(C) Sequencing chromatogram, the red box is the residue of target 1 (OsFAD2-T1), and the green box is the residue of target 2 (OsFAD2-T2)
58

• In plant, reducing or knocking out the expression of the FAD2 gene will reduce the
percentage of linoleic acid and subsequently will increase the level of oleic acid
• More than one sgRNA is recommended to target multiple sites in a single gene to
improve editing rates, especially in crops with low transformation efficiencies
• For efficient sgRNA target selection, highly specific nucleotide sequences to the genome
with GC content 50% and 65% were preferred to reduce off-target effects and produce
high editing efficiency
• Knocking out the two sgRNAs targeting a single gene caused large deletion of DNA
fragments between two target sites and subsequently generated homozygous mutant
allele T0 rice calli
• The oleic acid content was increased to 51.7% as compared to wild type ~ 32% via RNAi
technology and increased to approximately 80% via the CRISPR/Cas9 experiment
Conclusion
59

• Conservative site-specific recombination systems are involved in a wide variety of processes in
prokaryotes with functions in maintaining genome stability and transfer of mobile-genetic elements
• SSR enables the removal of extraneous DNA such as selectable markers, (i.e. antibiotic resistance
genes) from the genome as well as speeds the transition from laboratory manipulation to field
production
• SSR allows everything from single copy high throughput targeted integration, to sequential gene
stacking, to complete transgene removal from the pollen and/or seed
• Recombinase-mediated site-specific integration overcomes the complexity of DNA integration and
simplifies the structure, which, in turn, removes gene silencing triggers and creates a favourable
environment for stable gene expression
• Development of the CRISPR/Cas9 genome editing technology will have a revolutionary influence
on gene function research in plants and for crop genetic improvement
• CRISPR/Cas9-induced mutations are substantially equal to those generated by random mutagenesis,
but with much higher specificity and efficiency
• In addition, with segregation of genes between progeny during breeding, the transgenic T-DNA
region harbouring the Cas9 and sgRNA genes and the antibiotic-resistant marker genes can be
simply excluded, creating transgene-free gene-edited plant lines
Seminar’s Conclusion
60

• Further research on serine integrases for use in metabolic pathway construction and their
optimization, genetic circuits, logic gates, biocomputing, DNA-based nanodevices, genome
engineering, and control of heterologous protein or gene expression
• Some challenges that need to be overcome to improve the applicability of site-specific
recombinases include toxicity to the host cell, off-target activities, and inefficiency of some systems
• Different types of designer recombinases may address some of the issues pertaining to
recombinases
• Development of recombinases which have sites that are likely to be present in the natural genomic
sequence
• Development of CRISPR system for precise gene editing in plants, including DNA fragment knock-
in and gene replacement
• Cas9 variants should be modified to contain gene activation or repression domains can be used to
regulate the expression of targeted genes
• The CRISPR/Cas9 and similar systems may be adapted for many more novel applications, such as
RNA cleavage epigenomic regulation and chromatin imaging.
Future Thrust
61

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