This document provides an overview of RNA interference (RNAi) including its mechanism, applications, and methods for delivering small interfering RNA (siRNA). It discusses how dsRNA is processed by the enzyme Dicer into siRNAs which are incorporated into the RISC complex to degrade complementary mRNA. Viral vectors, liposomes, nanoparticles, and chemical modifications are described as methods used to deliver exogenous siRNAs. The document outlines both the therapeutic potential of RNAi and challenges associated with effective siRNA delivery.

1. Isfahan University of Medical Science, School of Pharmacy
Department of Clinical Biochemistry
June 25, 2012 1 Total slides : 51

2. RNA interference
Mechanism, Applications & Delivery Methods
Presented by:
A.N. Emami Razavi

3. O u t lin e s
Introduction
RNA silencing
Definition of RNA interference
Discovery of RNAi
Mechanism of RNA interference
Generation of small interfering RNA
Small interfering RNA delivery methods
Applications of RNA interference
Therapeutic applications
Other applications
Conclusion

4. In t r o d u c t io n
RN A i
(R N A In t e r f e r e n c e )

5. RNA silencing
Several terms are used to described RNA silencing;
usually there are three phenotypically different but
mechanistically similar phenomena:
3. Cosuppression or post-trascriptional gene silencing
(PTGS) in plants
5. Quelling in fungi
7. RNA interference in animal kingdom

6. Quelling:
The silencing in fungal system
Quelling came to light during attempts
to boost the production of an orange
pigment made by the gene al1 of the
fungus Neurospora crassa
N. crassa (al1+) transformed with
plasmid+al1
few transformants show albino
phenotype
al1-quelled strain had similar level of
unspliced al1 mRNA to wilde-type.
Native al1 mRNA was highly reduced
indicating that quelling and NOT the
rate of transcription affected the level
of mature mRNA in a homology-
dependent manner

7. RNAi:
Silencing in Cenorhabditis elegans
dsRNA administrated to
worms can permeate and
affect the entire body causing
a systemic RNA-interference
RNAi studies represents a
means of identifying partial or
complete loss-of-function
phenotypes, possibly leading
to the identification of gene
function.

8. Definition
RNA interference (RNAi) is a mechanism that inhibits
gene expression at the stage of translation or by
hindering the transcription of specific genes.
RNAi targets include RNA from viruses and
transposons.

9. Need for interference
Defense Mechanism
Defense against Infection by viruses, etc
As a defense mechanism to protect against transposons and
other insertional elements
Genome Wide Regulation
RNAi plays a role in regulating development and genome
maintenance.
30% of human genome regulated

10. PTGS in plants:
The discovery of Jorgensen and Napoli in
1990
They were trying to make petunias more purple
Overexpression of petunia gene
Entered homologous RNA
Expected:more pigments
Observed:white sectors
Cosuppression:
• Loss of mRNAs of both endo-and transgene

11. Andrew Fire Craig C. Mellow

12. Cenorhabditis elegans
RNAi can be induced in C. elegans in three simple ways:
Injection of dsRNA into the worm gonads
Soaking the worms in dsRNA solution
Feeding the worms engineered bacteria producing dsRNA

13. Discovery
Inject worms with dsRNA
corresponding to a gene
(important for muscle function)
involved in wiggling (unc-22)

14. Discovery
Inject worms with dsRNA
corresponding to a gene
(important for muscle function)
involved in wiggling (unc-22)
Conclusion: dsRNA triggers potent and specific gene silencing

15. RNAi vs Antisense
Effects of mex-3 RNA interference on
levels of the endogenous mRNA.
• Negative control showing lack of
staining in the absence of the
hybridization probe.
• Embryo from uninjected parent
showing normal pattern of
endogenous mex-3 RNA (purple
staining).
• Embryo from parent injected with
purified mex-3 antisense RNA.
• Embryo from a parent injected with
dsRNA corresponding to mex-3.
Injected antisense or dsRNA into C. elegans
dsRNA was more effective than ssRNA (antisense)
Effective even in tiny amounts
Inactivation was due to degradation of target mRNA

16. What is the secret of this little worms
success?
Simple organism
Similarity to other complicated animals
It has neurons,muscles, gut, …
Simple life cycle
Rapid growth
Produce about 300 progeny in 6 days
Eat bacteria
Suitable for genetic investigation

17. What is the secret of this little worms
success?

18. Nobel prize winners in the C. elegans
field Sidney Brenner
John Sulston
Robert Horvitz
Andrew Fire
Craig Mello

20. Happy ending
These results were thoroughly reproducible
RNAi was found to work in many species
RNAi became a powerful method that is changing the face of
biology
MicroRNAs were discovered (the next topic)
Andy and Craig win the 2006 Nobel Prize in Physiology or
Medicine!

21. What happened after this paper?

22. RNAi was found to work in many
diverse species
Fungi
Trypanosomes
Insects
Zebrafish
Mice

23. M e c h a n is m o f R N Ai
RN A i
(R N A In t e r f e r e n c e )

25. RNAi Overview
During RNAi Double-stranded RNAs cut into short
double-stranded RNAs, s(small) i(interfering) RNA's,
by an enzyme called Dicer. These then base pair to an
mRNA through a dsRNA-enzyme complex. This will
either lead to degradation of the mRNA strand
Highly specific process
Very potent activity
So far only been seen in eukaryotes
Evidence 30% of genome is regulated by RNAi

26. The Players In Interference
RNA
siRNA: dsRNA 21-22 nt.
miRNA: ssRNA 19-25nt. Encoded by non protein coding
genome
RISC:
RNA induced Silencing Complex, that cleaves mRNA
Enzymes
Dicer : produces 20-21 nt cleavages that initiate RNAi
Drosha : cleaves base hairpin in to form pre miRNA; which is
later processed by Dicer

27. siRNAs
Small interfering RNAs that have an integral role in the
phenomenon of RNA interference (RNAi), a form of post-
transcriptional gene silencing
RNAi: 21-25 nt fragments, which bind to the complementary
portion of the target mRNA and tag it for degradation
A single base pair difference between the siRNA template
and the target mRNA is enough to block the process.
Each strand of siRNA has:
a. 5’-phosphate termini
b. 3’-hydroxyl termini
c. 2/3-nucleotide 3’ overhangs

28. miRNA
Originate from capped & polyadenylated full length precursors
(pri-miRNA)
Hairpin precursor ~70 nt (pre-miRNA) Mature miRNA ~22 nt
(miRNA)

29. Difference between miRNA and siRNA
Function of both species is regulation of gene expression.
Difference is in where they originate.
siRNA originates with dsRNA.
siRNA is most commonly a response to foreign RNA (usually
viral) and is often 100% complementary to the target.
miRNA originates with ssRNA that forms a hairpin secondary
structure.
miRNA regulates post-transcriptional gene expression and is
often not 100% complementary to the target.
And also miRNA help to regulate gene expression, particularly
during induction of heterochromatin formation serves to
downregulate genes pre- transcriptionally (RNA induced
transcriptional silencing or RITS)
RITS

30. Dicer
RNase III-like dsRNA-specific ribonuclease
• Enzyme involved in the initiation of RNAi.
• It is able to digest dsRNA into uniformly
sized small RNAs (siRNA)
Dicer family proteins are ATP-
dependent nucleases.
Rnase III enzyme acts as a dimer
Loss of dicer→loss of silencing processing
in vitro
Dicer homologs exist in many organisms
including C.elegans, Drosphila, yeast and
humans (Dicer is a conserved protein)

31. Dicer’s domains
Dicer is a ribonuclease (Rnase III family) with 4 distinct domains:
domains
1 4 2 2 3
1. Amino-terminal helicase domain
2. Dual Rnase III motifs in the carboxy terminal segment
3. dsRNA binding domain
4. PAZ domain (110-130 amino-acid domain present in protein like
Argo, Piwi..);it is thought to be important for protein-protein
interaction

32. RISC
RISC is a large (~500-kDa)
RNA-multiprotein complex,
which triggers mRNA
degradation in response to
siRNA
Unwinding of double-
stranded siRNA by ATP
independent helicase.
The active components of an
RISC are endonucleases
called argonaute proteins
which cleave the target
mRNA strand.

33. Mechanism of RNA interference
• dsRNA are chopped into short
interfering RNAs (siRNA) by Dicer.
siRNA Dicer
2. The siRNA-Dicer complex recruits
additional components to form an
RNA-Induced Silencing Complex
(RISC). The siRNA unwinds.
RISC
3. The unwound siRNA base pairs with
complementary mRNA, thus
guiding the RNAi machinery to the
target mRNA.
4. The target mRNA is effectively
cleaved and subsequently
degraded – resulting in gene
silencing.
silencing

34. Exogenous dsRNA is detected and bound by an
effector protein, known as RDE-4 in C. elegans and
R2D2 in Drosophila, that stimulate dicer activity.This
protein only binds long dsRNAs.
These RNA-binding proteins then facilitate transfer of
cleaved siRNA to the RISC complex.

35. Mechanism of RNA interference

36. Mechanism of RNA interference

37. Amplification of siRNA

38. Illustration of miRNA processing

39. Another View

40. Summary of Players
Drosha and Pasha are part of the “Microprocessor”
protein complex (~600-650kDa)
Drosha and Dicer are RNase III enzymes
Pasha is a dsRNA binding protein
Exportin 5 is a member of the karyopherin
nucleocytoplasmic transport factors that requires Ran
and GTP
Argonautes are RNase H enzymes

42. G e n e r a t io n o f
s iR N A
RN A i
(R N A In t e r f e r e n c e )

43. Generation of small interference RNA

44. siRNA Expression Vectors

45. siRNA design
21-23nt
2-nt 3' overhangs ( UU overhangs )
G/C content: 30-50%.
No basepair mismatch
Synthesised siRNA should not target introns, the 5′and 3′-end
untranslated regions (UTR), and sequences within 75 bases of
the start codon (ATG).
BLAST : eliminate any target sequences with significant
homology to other coding sequences.

46. s iR N A d e l iv e r y
method s
RN A i
(R N A In t e r f e r e n c e )

47. Effective methods for the delivery of small RNA to allow a
sufficient silencing effect in the target organ(s) and/or cells are
yet to be developed.
In particular, toxicity and side effects of RNAi must be well
characterized and limited.
Therefore, careful design and selection of target sequence and
quantification of the effect on the expression of target protein and
mRNA are essential for success of gene interfering approaches.

48. High-pressure injection
“High-pressure injection” was the first strategy to demonstrate
injection
successful delivery of siRNA in vivo.
A large volume (1–2mL) of saline containing unmodified siRNA is
injected intravenously into the tail vein of mice within very short
time (in less than 7 sec), which presumably results in the siRNA
molecules being forced into several organs mainly the liver,
kidney and to a lesser degree the lung.
Certainly, such an approach seems to be impossible in human
subjects (1000 mL saline solution containing siRNA per 10 kg of
weight).

49. Electroporation
• Electroporation of small
RNA directly into target
tissues and organs has
also been developed to
successfully silence gene
function.

50. Problem
Delivery of siRNA to tissue is a problem both because:
The material must reach the target organ
And must also enter the cytoplasm of target cells.
RNA cannot penetrate cellular membranes, so systemic delivery
of siRNA is unlikely to be successful.
RNA is quickly degraded by RNAse activity in serum and even
siRNA chemically modified to be more stable has a half-life of
only a few hours at most.

51. Solution
For these reasons, other mechanisms to deliver siRNA to target
cells has been devised. These methods include:
Viral delivery
The use of liposomes or nanoparticles
Bacterial delivery
Chemical modification of siRNA to improve stability

52. Viral delivery
Viral delivery has been used extensively in gene therapy to
deliver DNA to target cells.
There are 5 main classes of viruses used in the delivery of
nucleotides to cells:
Retrovirus
Adenovirus
Lentivirus
Baculovirus
Adeno-associated-virus (AAV).

53. Retroviruses
Retroviruses were one of the first vectors used to transduct cells
with plasmids expressing hairpin-RNA constructs. Despite the
relative ease of use in vitro, use of the retrovirus in vivo has
safety concerns and significant limitations.
Retroviruses integrate their DNA into the host.s genomic DNA,
bringing with it, the risk of mutagenesis and carcinogenesis.
carcinogenesis
Another problem is that retroviral transduction is limited to
actively dividing cells, which means that the majority of
mammalian cells will not receive the siRNA.

54. Adenovirus
Adenoviral vectors are commonly used in gene therapy trials.
Since the adenovirus does not integrate DNA into the host.s
genome, the effects are short-lived, usually lost after several
cell divisions. For this reason, the adenovirus is used when a
short duration of action is sufficient or desirable, such as
tumor-targeting therapy.
Despite the lowered risk of insertional mutagenesis, the
adenovirus is associated with significant dose-dependent liver
toxicity that can severely limit therapy.
Another major disadvantage of adenoviral vectors is the
dependence on specific surface receptors on the target cell
which are often absent, rendering transduction impossible in
many cases.

55. Lentivirus
Lentiviral vectors are a subclass of retroviruses that lack the risk
of insertional mutagenesis and are able to transducer primary
and non-dividing cells.
Several studies have demonstrated the use of lentiviral vectors to
deliver RNAi to target cells.

56. Baculoviruses
Baculoviruses are insect viruses that can carry large quantities
of genetic information.
This may allow their use for combined RNAi therapy and gene
therapy.
Safety concerns are less prominent with these vectors since
the virus is unable to replicate or express proteins in
mammalian cells.

57. Adeno-associated viruses (AAV)
Adeno-associated viruses (AAV) are another possible vector for
siRNA.
These viruses do not appear to be pathogenic and can
transducer non-dividing cells.

58. Liposomes and nanoparticles
Liposomes and nanoparticles have been known as an alternative to
viral delivery systems.
Unmodified siRNA has a half-life of less than 1 hour in human plasma
and siRNA is rapidly excreted by the kidneys.
Liposomes and nanoparticles can act as envelopes to protect the
siRNA from metabolism and excretion, but can also carry specific
molecules designed to target the siRNA to specific tissue types.
Liposomes such as Lipofectamine, cationic DOTAP, neutral DOPC
have been used to carry siRNA into cells.
Nanoparticles such as the cationic polymer, polyethyleneimine (PEI)
have also been used to successfully deliver siRNA to target cells.

59. Bacterial delivery
Bacterial delivery using nonpathogenic bacteria has been used
to silence genes in a process known as transkingdom RNA
interference (tkRNAi).
Generally, the shRNA is produced in bacteria that invade and
release the RNA into eukaryotic cells (hence the term
transkingdom).
The bacteria can also be engineered to carry shRNA encoding
DNA plasmids.
The advantages of this system include:
Safety
Ability to control the vector using antibiotics

60. Chemical modification
Finally, chemical modification of siRNA has been used to
improve stability and prevent degradation by serum RNAase.
Importantly, these modifications must obviously not affect the
RNA interference activity of the siRNA.
One of the most common modifications is the use of locked
nucleic acid residues (LNA).
A methylene bridge connects the 4.C with the 2.O in LNA
residues. This modification increases the stability of
oligonucleotides in serum, without reducing the gene
silencing effect.

61. Side effects of gene silencing by small
RNA molecules
Unspecific silencing
Caused by the failure to identify similar sequences with only
few nt difference in other genes.
Activation of intracellular PKR and immune pathways that are
linked to toll-like receptor activation.
PKR ( protein kinase R) is activated by dsRNA longer than
30 nt, which subsequently induces the production of cytokines
of the IFN family. These IFNs ultimately promote inflammatory
responses.

62. Side effects of gene silencing…
High pressure injection” and electroporation can cause significant
injection
damage to the integrity of the normal tissues and organs and thus
preclude the utilisation in a clinical set-up.
Liposomes/cationic encapsulated siRNA may also be toxic to the
host and may cause severe host immune responses.
Other emerging strategies includes chemical modification of
siRNA molecules and encapsulated with different molecules are
still in their infancy and need to be thoroughly investigated before
used in therapeutic applications.

63. Ap p l ic a t io n s o f
R N Ai
RN A i
(R N A In t e r f e r e n c e )

64. Therapeutic uses of RNAi
Hematology (blood)
Oncology (cancer)
Stem cell biology
Infectious diseases

65. Hematology (blood)
Hematologic disorders result from
Loss of gene function
Mutant gene function
Absent gene function
RNAi
May be used to create models of disease processes
Could help to develop pharmacologic and genetic therapeutic
targets

66. Oncology (cancer)
Targeting of oncogenes
Dominant mutant oncogenes, amplified oncogenes, viral
oncogenes
Define role of signaling molecules in tumor-creation
Improvement efficacy of chemotherapy and
radiotherapy
Tumor regression through creation of potentially new
mode of chemotherapy

67. Stem cell biology
Mouse research
Knock out tumor-suppression gene in mouse embryonic
stem cell
Observe tumor phenotype
Positive correlation between extent of Trp 53
(suppression gene) inhibition and severity of disease

68. Infectious Diseases
Virus targeting
RNAi – inhibit cellular and viral factors of disease
RNA transcriptase is RNAi target
Inhibition of replication
Main goal
Render cells resistant to infectious organisms

69. Hepatitis C
Infects ~200 million people worldwide
Often fatal
2002, Anton McCaffrey and Mark Kay at Stanford
University
Injected "naked" RNA strands into the tail veins of
mice
RNAi treatment controlled the virus in mice

70. Silencing genes in HIV
AIM:
Silence the main structural protein in the virus, p24,
and the human protein CD4.
Hit the virus where it counts by eliminating a protein it
needs to reproduce or cause infections.

71. Respiratory infections
RSV, infects almost every child by the age of two
2005, Sailen Barik University of South Alabama
Short strands of "naked" RNA
Controlled the virus in mice
Clinical trials are ongoing

72. Macular degeneration
Macular degeneration is the leading
cause of adult blindness
Excess VEGF which leads to
sprouting of excess blood vessels
behind the retina & obscuring vision.
The new RNAi drugs shut down
genes that produce VEGF. The drug
can be injected directly into the eye
First clinical trial: 24 patients,
launched in 2004.
Two months after being injected with
the drug, 6 of the patients had
significantly clearer vision
Other patients' vision had at least
stabilized
More extensive trials are ongoing

73. Huntington’s disease
Ideal candidate for RNAi therapy
Disease caused by protein, that
affects more than 30,000 people
in the U.S. alone.
We would want to shut down the
expression of the gene coding for
the abberant protein
2004, Beverly Davidson and
colleagues at the University of
Iowa
Davidson treated mice with
Huntington's

74. Other uses of RNAi
Studying cell division
Testing Hypotheses of Gene Function
Target Validation
Pathway Analysis
• Gene Redundancy
Functional Screening

75. Studying cell division using RNAi
Genes involved in cell division identified by using RNAi
RNA interference (RNAi) used to assign functions of
genes involved in C. elegans cell division
133 genes identified. Only 11 previously identified

76. Testing Hypotheses of Gene Function
Array analysis and other methods for identifying differentially
expressed genes have created an enormous database of genes
and associated phenotypes.
In many cases, scientists make predictions about gene function
based on expression patterns in different samples. Other
predictions of mammalian gene function are developed using
homology searches with genes whose functions are known in
model organisms like Drosophila, C. elegans, and S. cerevisiae.
In many cases, testing the accuracy of these predictions can be
accomplished using siRNAs.

77. Testing Hypotheses of Gene Function:
……… still working
Al-Khalili et al treated myotubes with serum and showed that
increased glucose uptake correlated with increased cell-surface
content of glucose transporter (GLUT1). To confirm that glucose
transport depends on GLUT1 expression, cells were treated with
GLUT1 siRNA and were shown to have reduced levels of serum-
stimulated glucose transport.
Chen and Barritt used siRNAs to study the transient receptor
potential canonical 1 (TRPC1) gene. The TRPC1 gene was
thought to encode a non-selective cation channel activated by
depletion of cellular storage and/or an intracellular messenger.
When liver cells were treated with the TRPC1 siRNA, they
exhibited increased cell volume and decreased inflow of Ca2+,
Mn2+.

78. Target Validation
In its simplest form, drug development follows the path
of target identification → target validation →
therapeutic compound development → compound
testing in model systems → clinical trials.
siRNA are easy to use and highly specific, they
provide the ultimate tool for validation studies.
Reducing the expression of a potential therapeutic
target and determining if the desired phenotype results
provides confidence that an inhibitor of the same
target gene should have therapeutic value.

79. Target Validation:
… an interesting example
Filleur et al showed that the antiangiogenic molecule
thrombospondin-1 (TSP-1) could reduce vascularization and
delay tumor onset.
Over time, tumor cells producing active TSP1 began to form
exponentially growing tumors.
These tumors were composed of cells secreting unusually high
amounts of the angiogenic stimulator, vascular endothelial growth
factor (VEGF), which were sufficient to overcome the inhibitory
TSP1.
Treating tumor cells with a combination of TSP1 and a VEGF-
specific siRNA caused a striking reduction in cell proliferation.
This result suggested that using a combination of TSP1 and an
anti-VEGF compound would slow or eliminate tumor growth

80. Pathway Analysis
Reducing the expression of a single gene has implications on the
expression and activities of genes that are in the same
pathway(s).
Treating cells with an siRNA targeting a given gene and then
monitoring the expression of other genes using a microarray will
make it possible to identify genes that are associated with the
target gene.
Furthermore, a specific pathway can be dissected by treating
cells sequentially with siRNAs targeting the various genes in the
pathway and assaying which genes are affected. This will make it
possible to assign a position in the pathway for each gene.

81. Gene Redundancy
In many cases, eliminating the expression of a single gene in
higher eukaryotes can be tolerated even if that gene product
functions in a critical pathway. This is because many critical cell
functions are accomplished by more than one gene product.
When one gene product is eliminated, the redundant gene
product compensates to allow the cell or animal to survive.
Identifying redundant genes could be achieved by co-transfecting
siRNAs and assaying for a given phenotype.
Evaluating each of the candidate genes alone to ensure that they
only cause the cell cycle defect when reduced in combination
with the target gene would help pinpoint the most likely redundant
gene

82. Functional Screening
Libraries of siRNAs targeting broad collections of genes will
enable screening experiments to tie genes to cellular function.
To date, libraries with more than a couple of hundred siRNAs
have been limited to a few large research organizations.
Recognizing the benefits of siRNA libraries, Ambion is preparing
a collection of more than 1800 siRNAs targeting the known
human kinases.
There have been no published reports on the application of
siRNA libraries in screening experiments, but screens in
Drosophila and C. elegans using dsRNA libraries exemplify the
opportunities.

83. Biotechnology & Agriculture
RNA interference has been used for applications in
biotechnology, particularly in the engineering of food plants that
produce lower levels of natural plant toxins. Such techniques take
advantage of the stable and heritable RNAi phenotype in plant
stocks.
For example, cotton seeds are rich in dietary protein but naturally
contain the toxic terpenoid product gossypol, making them
unsuitable for human consumption.
RNAi has been used to produce cotton stocks whose seeds
contain reduced levels of delta-cadinene synthase, a key enzyme
in gossypol production, without affecting the enzyme's production
in other parts of the plant, where gossypol is important in
preventing damage from plant pests.

84. Biotechnology & Agriculture…
Similar efforts have been directed toward the reduction of the
cyanogenic natural product linamarin in cassava plants.
Although no plant products that use RNAi-based genetic
engineering have yet passed the experimental stage,
development efforts have successfully reduced the levels of
allergens in tomato plants and decreased the precursors of likely
carcinogens in tobacco plants.
Other plant traits that have been engineered in the laboratory
include the production of non-narcotic natural products by the
opium poppy, resistance to common plant viruses, and
fortification of plants such as tomatoes with dietary antioxidants.

85. C o n c l u s io n
RN A i
(R N A In t e r f e r e n c e )

86. D NA
RN A
P r o t e in

87. A
RN
D NA
n
ei
ot
Pr

88. Alzheimer’s
Asthma
Arthritis
Cancer
HIV
Hepatitis C & B
ALS
Muscular dystrophy
Cystic fibrosis
Smallpox
SARS
Macular degeneration
Influenza

89. RN A i

90. RNA interference characteristics
dsRNA needs to be directed against an exon, not an
intron in order to be effective
Homology of the dsRNA and the target gene/mRNA is
required
Targeted mRNA is lost (degraded) after RNAi
The effect is non-stoichiometric; small amounts of
dsRNA can wipe out an excess of mRNA (pointing to
an enzymatic mechanism)
ssRNA does not work as well as dsRNA

91. Advantage of RNAi
Downregulation of gene expression simplifies "knockout"
analysis.
Easier than use of antisense oligonucleotides. siRNA more
effective and sensitive at lower concentration.
Cost effective
High Specifity
middle region 9-14 are most sensitive
With siRNA, the researcher can simultaneously perform
experiments in any cell type of interest
Can be labelled
Ease of transfection by use of vector

92. Importance of RNAi
Powerful for analyzing unknown genes in sequenced genomes.
⇒ efforts are being undertaken to target every human gene via
siRNAs
Faster identification of gene function
Gene therapy: down-regulation of certain genes/ mutated alleles
Cancer treatments
knock-out of genes required for cell proliferation
knock-out of genes encoding key structural proteins
Agriculture

93. Th a n k y o u
A n y q u e s t io n ?