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1. GENE REPRESSION by miRNA and
SiRNA

2. RNA interference (RNAi)
• RNA interference (RNAi) refers to the mechanism of
gene silencing imparted by small noncoding RNAs with
varying levels of sequence complementarity to an
mRNA target.
• RNAi has evolved from a confusing experimental result
in plant and worm studies to a now well-characterized
process of gene expression control in multiple species.
• The recent awarding of the Nobel Prize in Medicine &
Physiology to researchers C. Mello and A. Fire for the
discovery of RNAi (Fire et al., 1998) solidifies its
standing among one of the major discoveries in cell
biology.

3. Structures of miRNA & siRNA
Both miRNAs and siRNAs are 21-23-nucleotide
single-stranded RNAs.
miRNAs bind to the 3' UTR regions of
complementary mRNAs via imperfect base-pairing.
Thus they often can inhibit translation of more than
one mRNA.
siRNAs hybridize perfectly without any mismatches
to the coding region of their target mRNAs
Thus they typically regulate only a single mRNA
species.

5. Mechanism of Action of mi- and siRNAs
RNA Pol II transcribes pri-miRNA transcripts that are
partially double-helical.
The pri-miRNA is processed to a shorter ~ 70 nt pre-
miRNA that is then transported to the cytoplasm.
The pre-miRNA, which folds into a hairpin structure,
is bound by a protein complex containing the
enzyme known as Dicer.
Dicer cleaves the molecule producing a 21-23-nt
double stranded miRNA.
Finally, one of the strands is bound by a protein
complex known as the RISC complex (RNA-induced
silencing complex).

6. • The RISC/miRNA complex subsequently binds to
the 3’ UTR of a target mRNA leading to its
sequestration away from ribosomes.
• siRNAs are generated in the Dicer reaction from
double-helical RNAs introduced into cells or
produced from cleavage of viral RNAs.
• They also are bound by the RISC complex.
However, RISC/siRNA complexes bind to the
coding region of a target mRNA, and ultimately
the target mRNA is cleaved at a site within the
perfect siRNA-mRNA duplex

8. CRISPR: A game-changing genetic
engineering technique
• What is CRISPR
• CRISPR is an acronym for Clustered Regularly Interspaced Short
Palindromic Repeat.
• This name refers to the unique organization of short, partially
palindromic repeated DNA sequences found in the genomes of
bacteria and other microorganisms.
• CRISPR sequences are a crucial component of the immune systems
• The functions of CRISPR (Clustered Regularly Interspaced Short
Palindromic Repeats) and CRISPR-associated (Cas) genes are
essential in adaptive immunity in select bacteria and archaea,
enabling the organisms to respond to and eliminate invading
genetic material.
• These repeats were initially discovered in the 1980s in E. coli but
their function wasn’t confirmed until 2007 by Barrangou and
colleagues, who demonstrated that S. thermophilus can acquire
resistance against a bacteriophage by integrating a genome
fragment of an infectious virus into its CRISPR locus

9. • Three types of CRISPR mechanisms have been
identified, of which type II is the most studied.
In this case, invading DNA from viruses or
plasmids is cut into small fragments and
incorporated into a CRISPR locus amidst a
series of short repeats (around 20 bps).
• The loci are transcribed, and transcripts are
then processed to generate small RNAs (crRNA
– CRISPR RNA), which are used to guide
effector endonucleases that target invading
DNA based on sequence complementarity

10. In the acquisition phase, foreign DNA is incorporated into the bacterial genome at
the CRISPR loci.
CRISPR loci is then transcribed and processed into crRNA during crRNA biogenesis.
During interference, Cas9 endonuclease complexed with a crRNA and separate
tracrRNA cleaves foreign DNA containing a 20-nucleotide crRNA complementary
sequence adjacent to the PAM sequence. (Figure not drawn to scale.)

11. How CRISPR/Cas9 works as immune system in bacteria.
When the invader (plasmid or virus) enters bacteria it directs a nuclease called Cas2 to
snip a short sequence of the viral genome (spacer) and insert it between two repeats in
its CRISPR locus .
When this invader type come again the bacteria transcribe its spacer to generate crRNA
which will be matured by tracrRNA(Trans activating cr RNA)
Both types of RNA associated with Cas9 will be directed to the invader genome to cleave
it (using Cas9) after recognizing it (using crRNA)

12. • Breast cancer is a leading cause of death in females worldwide .
• Breast cancer rate is rising worldwide with an expansion in forceful
neoplasia in women. Around half of the breast malignancy cases
and 60% of the deaths are happening in developing countries.
• There is an extensive distinction in breast cancer rate among
Hispanic, Caucasian and Asian women, with Caucasian women
being the most astounding and Asian women being the least
• Breast cancer research is a hot area in which CRISPR technology is
in the core
• CRISPR/Cas9 takes its place as an essential, efficient, and
straightforward tool for cancer research, especially breast cancer
• This tool provides a means to insert, correct, and remove the faulty
genes in a precise manner. Some limitations are facing applying this
technology in all types of cancer; however, advancements are
taking place