Gene silencing and Edit for life science

1. Presented by:
Mesele Tilahun
(Integ.1st year)
Advisor: MoonJae Sun
GENE
SILENCING
and
EDITING
Oct 27, 2020
KRIBB, UST , S.Korea
RNA biology presentation

2. Contents
GENE SILENCING
-RNAI ( Si RNA vs miRNA)
- Ribozyme
GENE EDITING
- CRISPR/CAS 9
APPLICATION
SUMMARY

3. GENE SILENCING
Gene silencing is the regulation of gene
expression
it occur either transcription or translation level.
 methods used to silence genes
-RNAi (miRNA and siRNA),
- Ribozymes
- CRISPR

5. Gene silencing methods used in research
• Antisense oligonucleotides :
-bind to complementary target molecules
• Ribozymes :
-catalytic activity inhibit gene expression
- not only bind, but can also cleave, their target
RNA.

6. Early Findings Of GS In Plants
First discovered in plants by R. Jorgensen in1990
 When Jorgensen introduced a re-engineered
gene into petunia that had a lot of homology
with an endogenous petunia gene, both genes
became suppressed!
(Stam et al., 1997)
Aim: to increase pigment, Result: loss of pigmentation in segments

8. RNA interference (RNAi)
• RNA interference (RNAi) is a biological process
in which RNA molecules inhibit gene expression or
translation, by neutralizing targeted mRNA
molecules
• RNAi was known by other names,
=>co-suppression,
=>post-transcriptional gene silencing (PTGS),
=> quelling
• RNAi is precise, efficient, stable and better than
antisense technology for gene suppression

9. RNAi
• Andrew Fire and Craig C. Mello (2006) Nobel Prize for
their work on RNA interference in the Caenorhabditis
elegans
• Two types of small ribonucleic acid (RNA) molecules
– microRNA (miRNA)
-small interfering RNA (siRNA)
• small RNAs degrade messenger RNA (mRNA) molecules
(a) translation, via post-transcriptional gene silencing
(b) transcription, via the pre-transcriptional silencing
• enzyme complex catalyzes DNA methylation at genomic
positions complementary to complexed siRNA or miRNA.

10. (Courdavault et al. 2020)

11. Endogenous triggers of RNAi pathway
https://www.ncbi.nlm.nih.gov/probe/docs/techrnai/
-include foreign DNA or dsRNA of viral origin, aberrant transcripts from repetitive
sequences in the genome such as transposons, and pre-microRNA (miRNA).
In plants, RNAi ==> (VIGS), suggesting an important role in pathogen resistance.

12. A simplified model for the RNAi pathway
- Is based on two steps, each involving ribonuclease enzyme.
1st step, the trigger RNA (dsRNA / miRNA primary transcript) is processed into an siRNA by the RNase II
enzymes Dicer and Drosha. 2nd step, siRNAs are loaded into the effector complex (RISC).  unwound
during RISC assembly and ss RNA hybridizes with mRNA target. Gene silencing is a result of
nucleolytic degradation of the targeted mRNA by the RNase H enzyme Argonaute (Slicer).

13. the enzyme involved in RNAi is Dicer
• cleaves long dsRNA into short ds fragments of ~21
ntds siRNAs
• Each siRNA is unwound into two ssRNAs, the
passenger strand and the guide strand
• The passenger strand is degraded and the guide
strand is incorporated into the RNA-induced
silencing complex (RISC)
• when the guide strand pairs with a complementary
seq in mRNA and induces cleavage by Argonaute2
(Ago2), the catalytic component of the RISC.

14. siRNA vs miRNA
Which one is coding RNAs ? siRNA or miRNA
Both does not encode a protein but important
in gene regulation
short RNA duplexes that target mRNA(s) to
produce a gene silencing effect

15. ### Si RNA MiRNA
Origins originates with dsRNA Originates with ssRNA that forms a hairpin
secondary structure.
Response and
complementarity
Mostly response to foreign RNA
(usually viral) and is often 100%
complementary to the target.
regulates post-transcriptional gene expression and
is often not 100% complementary to the target
mRNA target inhibit the expression of one
specific target mRNA
regulate the expression of multiple mRNAs
Prior to Dicer
processing
ds RNA that contains 30 to over
100 ntds
Precursor miRNA (pre-miRNA) that contains 70-100
ntds with interspersed mismatches and hairpin
structure
Structure 21-23 ntd RNA duplex with 2
nucleotides 3'overhang
19-25 ntd RNA duplex with 2 nucleotides
3'overhang
Complementary Fully complementary to mRNA Partially complementary to mRNA, typically
targeting the 3' UTR region of mRNA
Mechanism of gene
regulation
Endonucleolytic cleavage of mRNA Translational repression Degradation of mRNA
Endonucleolytic cleavage of mRNA (rare, only
when there is a high level of complementary
between miRNA and mRNA)
Comparison of general properties between siRNA and miRNA

16. Gene silencing mechanisms of siRNA and miRNA (Lam et al., 2015)

17. (a) siRNA is usually fully complementary to the coding region of its target mRNA;
(b) miRNA is partially complementary to its target mRNA.
Complementary binding usually occurs at the seed region (nucleotides (nt) 2–7 of
the 5’ end) of miRNA and the 3’ UTR of the target mRNA.
Target recognition by siRNA and miRNA.

18. Antisense RNA inhibits protein synthesis by blocking Translation
Ribozymes

19. Ribozymes
• Ribozymes are catalytic RNA molecules used in
gene silencing
• work by cleaving mRNA molecules.

20. https://www.ncbi.nlm.nih.gov/probe/docs/applsilencing/

21. Technological applications
1. Gene knockdown: to study the function of
genes
2. Functional genomics: for genomic mapping
3. Medicine: use siRNA to treat cancer by
silencing genes differentially up regulated
4. Biotechnology : Flavr Savr tomato and two
cultivars of ring spot-resistant papaya, were
originally developed using antisense technology.

23.  GABA accumulates in many plant species in response to
environmental stress.
Authors studied the 3 metabolic pathway controlling steps of GABA
Through VIGS approach
- glutamate decarboxylases (SlGADs),
- GABA transaminases (SlGABA-Ts) and
- succinic semi aldehyde dehydrogenase (SlSSADH),
(Bao et al., 2015)

24. The silecing of
-SlGADs- GABA biosynthetic
genes
-SlGABA-Ts- GABA
catabolic genes
accumulate (ROS) than
control plants salt sensitivity.
-SlSSADH-silenced plants accumulated
higher level of ROS under normal conditions
- less sensitive to salt stress
These results suggest that GABA shunt is involved in salt
tolerance of tomato, probably by affecting the homeostasis of
metabolites such as succinate and γ-hydroxy butyrate and
subsequent ROS accumulation under salt stress.

25. Genome editing is a type of genetic engineering in which DNA is inserted, replaced,
or removed from a genome using artificially engineered nucleases, or "molecular
scissors”.
Genome editing tools :
1) Zinc finger nucleases (ZFNs):
2) Transcription activator–like effector nucleases (TALENs):
3) CRISPR-Clustered Regularly Interspaced Short Palindromic Repeats
Genome Editing for gene knockout

26. Mechanism of CRISPR/Cas9 complex
Editing use engineered nucleases, or "molecular
scissors".
 Creating Nuclease induced DSBs (Double stranded
breaks) .
 Repairing DSBs in either one of the two pathways.
»NHEJ
[Non-homologous End Joining]
»HDR repair
[Homology Directed Repair]
 resulting in targeted mutations ('edits').
(Vats et al., 2019), (Moreira et al., 2020)

27. Pathways for repair of DSBs induced by genome editing tools. NHEJ & HR in the presence
of a donor template
(Davis & Ph, 2013)

28. HDR
• is a DNA molecule that uses HDR (HR) to transfer
genetic information to a chromosome, and is
widely used for knockouts, mutagenesis, gene
tagging, promoter swapping, and trans genesis.
• is initiated by CRISPR- or TALEN-mediated site-
specific DSBs.
• Cells repair DSBs by two mechanisms (Fig):
• 1st is NHEJ, which causes small insertions or
deletions (“indels”) without a homologous
template  causes frame shift mutations and is
used to knock genes out.

29. HDR
• 2nd major pathway used for DSB repair is HDR.
Compared with NHEJ,
• HDR is relatively error-free Besides sister chromatids,
exogenous DNA used as an HDR template.
• consists of left and right “arms” identical to sequences
flanking the DSB (Figure 1).
• Recombination “copies” information from the donor
and “pastes” it to the chromosome.
• Researchers can place virtually any DNA seq b/n the
arms—Selectable marker, GFP, etc.—and
• HDR will permit these additional sequences to be
copied, or “knocked in” to the chromosome.

30. CRISPR
family of DNA sequences found within the genomes
of prokaryotic organisms (bacteria and archaea)
are derived from DNA fragments from viruses that
have previously infected the prokaryote and
are used to detect and destroy DNA from similar
viruses during subsequent infections

31. Cas9 (CRISPR-associated protein 9)
is an enzyme that uses CRISPR sequences as a
guide to recognize and cleave specific strands
of DNA that are complementary to the CRISPR
sequence.
 CRISPR/Cas9 can be used to edit genes within
organisms.
• is an RNA guided DNA endonucleases enzyme.
(Wang et al., 2019)

32. (Kim et al., 2019)

34. Summary
Method Knock down Knock out Change
genetic code
Change
expression
level
Clone
isolation
required
RNAi (shRNA,
siRNA)
✓ ✓
Genome editing
(TALEN, CRISPR)
✓ ✓ ✓ ✓
Comparisons between RNAi and genome editing