Gene silencing is a method of regulating gene expression to reduce or prevent the expression of certain genes. It can occur during transcription or translation and is often used in research. Methods of gene silencing like RNAi, CRISPR, and siRNA are being researched as potential therapeutics for diseases. Gene silencing reduces gene expression by at least 70% but does not eliminate it entirely, allowing study of essential genes. RNA interference is a common method of gene silencing that uses small interfering RNAs or microRNAs to regulate gene expression.

1. [GENE SILENCING AND RNAi]
Submitted by:
Shiney Chatak
(J-20-D-399-A)

2. Gene silencing is the regulation of gene expression in a cell to prevent the
expression of a certain gene. It is a method to silence, suppress or reduce the
expression of certain genes or genes of our interest by genetic engineering
techniques. Gene silencing can occur during
either transcription or translation and is often used in research. In particular,
methods used to silence genes are being increasingly used to
produce therapeutics to combat cancer and other diseases, such as infectious
diseases and neurodegenerative disorders.
Gene silencing is often considered the same as gene knockdown. When genes
are silenced, their expression is reduced. In contrast, when genes are knocked
out, they are completely erased from the organism's genome and, thus, have no
expression. Gene silencing is considered a gene knockdown mechanism since
the methods used to silence genes, such as RNAi, CRISPR, or siRNA, generally
reduce the expression of a gene by at least 70% but do not completely eliminate
it. Methods using gene silencing are often considered better than gene
knockouts since they allow researchers to study essential genes that are required
for the animal models to survive and cannot be removed. In addition, they
provide a more complete view on the development of diseases since diseases are
generally associated with genes that have a reduced expression.
Types of gene silencing:
RNA interference-mediated gene silencing:
In the RNAi mechanism, either siRNA or miRNA governs the process of gene
silencing. Here the siRNA is the small interfering RNA while the miRNA is
microRNA, both are non-coding RNAs that regulate gene expression through
different mechanisms.
Transcriptional gene silencing
Epigenetic factors such as methylation, acetylation, histone modifications and
chromatin remodelling also make genes inactive.
DNA methylation is the most popular mechanism and known to us for gene
silencing. The enzyme SAM adds methyl groups on the CpG region of DNA
and makes it inactive. During histone modification, histones like H2A, H2B, H3
and H4 make a complex with DNA (known as nucleosome), converts it in the
heterochromatin region and makes it transcriptionally inactive. Chromatin

3. remodelling is also one of them that does do the same. All these epigenetic
factors help DNA or genes to pack so tightly thus enzymes and transcriptional
factors can’t access it. They can’t form protein, resultantly.
Transposons in gene silencing:
The transposons are the mobile genetic elements that can move from one place
to another place in a genome. DNA transposons and retrotransposons are two
types of transposon systems present commonly in prokaryotes and eukaryotes,
respectively. Transposable elements are the natural genetic elements involved in
gene silencing. The elements jump from one location to the active gene where it
inserts in it. The active gene now has some extra gene sequence that is not a part
of it actually, hence it can’t perform translation. Sleeping beauty transposon
system is now used in genetic engineering to manipulate gene expression.
Antisense oligonucleotides:
A method in which by designing some short-nucleotide sequences specific to
the mRNA we wish to silence, to make a gene inactive. This method is known
as antisense oligonucleotides. The present method was first reported by Paul
Zamecnik and Mary Stephenson in 1978. The complementary antisense
nucleotides hybridize to its complementary region on mRNA and either cleave
it using the RNase or blocks the translation by some other means.
In both cases, mRNA can’t form a protein. The method is traditionally known
as antisense RNA technology.
CRISPR-CAS9 gene silencing:
CRISPR-CAS9 is a great tool for gene editing. In normal CAS9 activity,
the single-stranded guided RNA recognizes the nuclease CAS9 and guides it to
cleave the nucleic acid sequence and hence the gene can’t form protein.
Scientists have developed a special type of CAS9 nuclease that can bind to the
target nucleic acid or gene but can’t cut it, consequently, the polymerase and
other transcriptional factors can’t identify the sequence. Protein cannot form
from it, resultantly.

4. Besides the antisense oligonucleotide techniques and altered CRISPR-CAS9,
other gene silencing methods naturally occur in prokaryotes and eukaryotes as
their defence system to protect a cell. CRISPR-Cas9 technology behaves like a
cut-and-paste mechanism on DNA strands that contain genetic information.
Specific location of the genetic codes that need to be changed, or “edited”, is
identified on the DNA strand, and then, using the Cas9 protein, which acts like
a pair of scissors, that location is cut off from the strand. A DNA strand, when
broken, has a natural tendency to repair itself. Scientists intervene during this
auto-repair process, supplying the desired sequence of genetic codes that binds
itself with the broken DNA strand.

5. In addition to this, in mammals, the gene silencing regulates the cell cycles and
cell division. The RNA silencing or suppressing has an important role in the
metabolism of cells.
Applications of gene silencing:
Gene silencing has a tremendous role in genetic engineering and transgenic
construction. In the plant genetics various economically important plants can be
constructed using the present method.
In the medical field, the gene silencing technique is used to study genes
associated with cancer, infectious disorders and other genetic
disorders. Overexpression of some genes causes cancer, which is silenced by
the shRNA and miRNA mediated technique.
The gene silencing is also used in plant genetics for creating genetically
modified organisms or plants that are economically important. Genetically
engineered plant species that produce less toxin are now constructed using the
present RNA interference technique.
The siRNA mediated gene silencing is used in treating infectious diseases like
HIV. Here the viral RNA gene is targeted using the siRNA which binds to it and
makes it inactive transcriptionally. This technique is now under the trial phase
for HIV and hepatitis infection, although results are unambiguous.
Scientists are now applying the gene silencing method to treat diseases like
asthma, cystic fibrosis, chronic obstructive pulmonary disease, hepatitis B,
hepatitis C, chronic myeloid leukaemia and neurodegenerative disorders.
It is used in agribiotechnology, microbiology, food processing technology and
in other science fields for various applications.
Gene knockdown vs gene silencing:
The gene knockdown and gene silencing are two different techniques.
In the gene knockdown we are stopping our genes from expressing, means, we
are disrupting the genes normal function and protein can’t form. This technique
is used to stop the production of faulty protein but can’t minimize gene
expression.

6. While in gene silencing technique, we are making a gene inactive to some
extent, means, we are not inactivating it entirely. So overall the expression of a
gene or amount of protein formation reduces but won’t stop!
RNAi
RNAi or Post-Transcriptional Gene Silencing (PTGS) is a gene silencing
technology that inhibits protein synthesis in target cells using double-stranded
RNA. It is mechanism that inhibits gene expression at the stage of translation
or by hindering the transcription of specific genes. It is a conserved biological
response to double-stranded RNA that mediates resistance to both endogenous
parasitic and exogenous pathogenic nucleic acids, and regulates the expression
of protein-coding genes. This natural mechanism for sequence-specific gene
silencing.
History
R. Jorgensen and his colleagues identified a novel mechanism of post-
transcriptional gene silencing in Petunia. They were attempting to introduce a
chalcone synthase gene under a strong promoter to deepen the purple colour
of Petunia flowers; however, instead
of getting a stronger purple colour
flower they observed that most flowers
lost their colour. Thus, they observed
diminished expression of both the
homologous endogenous gene and the
exogenously introduced transgenic
copy of the gene and termed the
phenomenon as co-suppression.
In 1995, Guo and Kempheus attempted
to knock down the expression of PAR-
1 gene by antisense RNA
in Ceanorhabdites elegans; they
observed a similar loss of gene
expression with sense RNA controls as
well. At that time, they could not
explain the mechanistic basis of such
an observation. In 1998, Andrew Fire,
Craig C. Mello, and their colleagues demonstrated efficient and specific
interference of gene expression by introducing double-stranded RNA in the

7. nematode C. elegans. The genetic interference was genetically heritable and
was stronger than the antisense strategy. This novel phenomenon was termed as
RNA interference or RNAi by Fire and colleagues.
Requirements of RNAi
• Small interfering RNAs (siRNAs)
Small interfering RNAs are 21–23-nt-long double-stranded RNA
molecules with 2–3-nt overhangs at the 3′ termini. siRNAs are normally
generated, as mentioned in the above sections, by the cleavage of long
double-stranded RNAs by RNase III (Dicer). siRNAs must be
phosphorylated at the 5′ termini by endogenous kinases to enter into the
RISC complex.
• Micro RNAs (miRNAs)
miRNAs are 19–25-nt small RNA species produced by Dicer-mediated
cleavage of endogenous ~70-nt noncoding stem-loop precursors. The
miRNAs, while allowing mismatches, can either repress the target mRNA
translation (mostly in mammals) or facilitate mRNA destruction (mostly
in plants). miRNAs lin-4 and let-7 were the first ones to be identified
in C. elegans. So far, about 2000 different miRNAs have been identified
in plants, animals, and lower species.
Comparative account of siRNA and miRNA

8. • RNA-Induced Silencing Complex (RISC)
RISC is a ribonucleoprotein complex that fragments mRNAs through the
production of a sequence-specific nuclease.
• Dicer
Dicer was first characterized and defined in Drosophila by Bernstein et
al. Dicer belongs to the RNase III-class and assists in ATP-dependent
siRNA generation from long dsRNAs. Dicer is a large (~220-kDa) multi-
modular protein that acts as an antiparallel dimer.
Mechanism
RNAi-mediated gene silencing is executed by siRNAs. The process of
silencing begins with the cleavage of long dsRNAs into 21–25 -nt
fragments of siRNAs in cytoplasm. The process is catalyzed by Dicer
enzyme. These siRNAs are inserted into multiprotein silencing complex,
which is known as RNA-induced silencing complex (RISC). Subsequent
unwinding of siRNA duplex, in turn, leads to active confirmation of
RISC complex (RISC*). Next, target mRNA (mRNA to be degraded) is
recognized by antisense RNA, which signals RISC complex for the
endonucleolytic degradation of the homologous mRNA. Tuschl and his
colleagues have defined the directionality of dsRNA processing and the
target RNA cleavage sites. According to their results, target mRNA is
cleaved in the centre of the region that is recognized by complimentary
guide siRNA, which is 10–12 -nt away from the 5′ terminus of siRNA.
The RNAi process is completed by the last step of siRNA molecule
amplification. It is well established that the next generation of siRNAs is
derived from the priming on the target mRNA by RNA-dependent RNA
polymerase (RdRp) enzyme by existing siRNAs. The second generation
of siRNAs is effective in inducing a secondary RNA interference that is
defined as transitive RNAi. The transitive RNAi causes a systemic
genetic interference in plants and C. elegans. Interestingly, transitive and
systemic RNAi is absent in Drosophila and mammals owing to the lack
of RdRp in both organisms.

9. 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).
Advantages of RNAi
• Downregulation of gene expression simplifies "knockout" analysis. RNA
interference pathway is often exploited in experimental biology to study
the function of genes in cell culture and in vivo in model organisms.
Double-stranded RNA is synthesized with a sequence complementary to
a gene of interest and introduced into a cell or organism, where it is
recognized as exogenous genetic material and activates the RNAi
pathway. Using this mechanism, researchers can cause a drastic decrease
in the expression of a targeted gene. Studying the effects of this decrease
can show the physiological role of the gene product. Since RNAi may not
totally abolish expression of the gene, this technique is sometimes
referred as a "knockdown", to distinguish it from "knockout" procedures
in which expression of a gene is entirely eliminated.
• Easier than use of antisense oligonucleotides

10. • siRNA more effective and sensitive at lower concentration
• Cost effective
• Highly Specific, 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
Importance
• Powerful for analysing 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
Use of RNAi in agriculture
Scientists are now using the RNAi and antisense RNA technology in crop
improvement. Novel plant traits and disease-resistant species of plants are
being developed using the present technology. Moreover, it is also used
in pest control and yield improvement. Flvr Savr tomato, decaffeinated
coffee and nicotine-free tobacco are some of the best examples of plant
species developed using RNAi technology.
• RNAi for Disease and Pathogen Resistance
Gene silencing was first used to develop plant
varieties resistant to viruses. Engineered antiviral
strategies in plants mimic natural RNA silencing
mechanisms. This was first demonstrated when
scientists developed Potato virus Y- resistant
plants expressing RNA transcripts of a viral
proteinase gene. Immunity has since been shown to other viruses such as
the Cucumber and Tobacco Mosaic Virus, Tomato Spotted Wilt Virus,
Bean Golden Mosaic Virus, Banana Bract Mosaic Virus, and Rice
Tungro Bacilliform Virus among many others.
In addition, plants can also be modified to produce dsRNAs that silence
essential genes in insect pests and parasitic nematodes. This approach

11. was used to develop root-knot nematode, corn rootworm and cotton
bollworm resistant varieties.
• RNAi for Male Sterility
RNAi has also been used to generate male sterility, which is valuable in
the hybrid seed industry. Genes that are expressed solely in tissues
involved in pollen production can be targeted through RNAi. For
instance, scientists have developed male sterile tobacco lines by
inhibiting the expression of TA29, a gene necessary for pollen
development. RNAi was also used to disrupt the expression of Msh1 in
tobacco and tomato resulting to rearrangements in the mitochondrial
DNA associated with naturally occurring cytoplasmic male sterility.
• RNAi and Plant Functional Genomics
A major challenge in the post-genomic era of plant biology is to
determine the functions of all genes in the plant genome. Compared to
other techniques, RNAi offers specificity and efficacy in silencing
members of a gene or multiple gene family. In addition, the expression of
dsRNAs with inducible promoters can control the extent and timing of
gene silencing, such that essential genes are only silenced at chosen
growth stages or plant organs.
There are several ways of activating the RNAi pathway in plants. The
various RNAi techniques have advantages and disadvantages with respect
to how persistent their effects are and the range of plants to which they
can be applied. These include the use of hairpin RNA-expressing vectors,
particle bombardment, Agrobacterium- mediated transformation and
virus-induced gene silencing (VIGS).
• Engineering Plant Metabolic Pathways through RNAi
RNAi has been used to modify plant metabolic pathways to enhance
nutrient content and reduced toxin production. The technique takes
advantage of the heritable and stable RNAi phenotypes in plants.
Prospects for RNAi in relation to agriculture
With RNAi, it would be possible to target multiple genes for silencing
using a thoroughly-designed single transformation construct. Moreover,
RNAi can also provide broad-spectrum resistance against pathogens with
high degree of variability, like viruses. Recent studies have hinted
possible roles of RNAi-related processes in plant stress adaptation.
Although, much progress has been made on the field of RNAi over the
past few years, the full potential of RNAi for crop improvement remains

12. to be realized. The complexities of RNAi pathway, the molecular
machineries, and how it relates to plant development are still to be
elucidated.
Examples of novel plant traits engineered through RNAi.