A modern way of gene silencing.

1. RNA interference (RNAi):
A therapeutic strategy for aquaculture
Presented By – MD. IDRISH RAJA KHAN
M.F.Sc. (AAH – 01/15)
COF, CAU (I), TRIPURA
Master’s seminar onMaster’s seminar on

2. INTRODUCTIONINTRODUCTION
• RNAi, a natural mechanism for post-transcriptional
silencing of homologous genes by dsRNA resulting
in inhibition of translation (Fire et al., 1998).
• In 1998, Andrew Fire and Craig Mello were the
first to reveal the induction of RNAi in the
nematode worm Caenorhabditis elegans.
• Later on for the year 2006 they were awarded by
Nobel Prize in the field of Physiology or Medicine.
• RNAi, a natural mechanism for post-transcriptional
silencing of homologous genes by dsRNA resulting
in inhibition of translation (Fire et al., 1998).
• In 1998, Andrew Fire and Craig Mello were the
first to reveal the induction of RNAi in the
nematode worm Caenorhabditis elegans.
• Later on for the year 2006 they were awarded by
Nobel Prize in the field of Physiology or Medicine.

3. How RNAi Works ?How RNAi Works ?
Two-step mechanism for RNAi:
A. INITIATION STEP
1. The entry of long dSRNA, triggers the RNAi
pathway of cells. This results in the activation of
the enzyme Dicer.
2. Dicer cleaves the dsRNA into short, 20-21
basepairs long, fragments, called small interfering
RNA (siRNA). These small molecules have a
characteristic 2 nucleotide 3’ overhang.
Two-step mechanism for RNAi:
A. INITIATION STEP
1. The entry of long dSRNA, triggers the RNAi
pathway of cells. This results in the activation of
the enzyme Dicer.
2. Dicer cleaves the dsRNA into short, 20-21
basepairs long, fragments, called small interfering
RNA (siRNA). These small molecules have a
characteristic 2 nucleotide 3’ overhang.

4. B. EFFECTOR STAGE
1. Short RNA duplexes are incorporated into a
multimeric protein complex, known as RNA-induced
silencing complex (RISC), which contains an
Argonaute (Ago) protein as one of its main
components.
2. RISC binds and unwinds the siRNAs into single-
stranded molecules. The sense strand is released
and the antisense strand remains bound to RISC,
serving as guide to select fully complementary
mRNA (seed) substrates for degradation.
B. EFFECTOR STAGE
1. Short RNA duplexes are incorporated into a
multimeric protein complex, known as RNA-induced
silencing complex (RISC), which contains an
Argonaute (Ago) protein as one of its main
components.
2. RISC binds and unwinds the siRNAs into single-
stranded molecules. The sense strand is released
and the antisense strand remains bound to RISC,
serving as guide to select fully complementary
mRNA (seed) substrates for degradation.

5. Fig. RNA interference mechanism Source – www.nature.com

6. DEVELOPING THERAPEUTIC RNAiDEVELOPING THERAPEUTIC RNAi
There are several stages in developing a RNAi based
therapy for viral diseases –
1. Target gene
2. Target sequence
3. Effective design
4. Therapeutic molecules - siRNA, miRNA
5. Delivery mode
Immersion, feeding, hydrodynamic i.v. injection,
electroporation, cholesterol conjugates, cationic delivery
systems, nano delivery systems etc.
There are several stages in developing a RNAi based
therapy for viral diseases –
1. Target gene
2. Target sequence
3. Effective design
4. Therapeutic molecules - siRNA, miRNA
5. Delivery mode
Immersion, feeding, hydrodynamic i.v. injection,
electroporation, cholesterol conjugates, cationic delivery
systems, nano delivery systems etc.

7. SOME SUCCESSFULL IMPLEMENTATION OF RNAi IN
AQUACULTURE
SOME SUCCESSFULL IMPLEMENTATION OF RNAi IN
AQUACULTURE
Shrimps Gene
construct
Protection
Studies against
Gene References
Penaeus
monodon
siRNA WSSV Vp28, vp15 Westenberg
et al. (2005)
Litopenaeus
vannamei
siRNA WSSV DNA pol, Rr2,
ORF252, vp28
Robalino
et al. (2005)
P. monodon siRNA YHV Protease Yodmuang
et al. (2006)
L. vannamei siRNA TSV DNA pol, Rr2, Wu et al.
(2007)
P. chinensis siRNA WSSV vp28, vp281,
Protein kinase
Kim et al.
(2007)
Metapenaeus
japonicus
siRNA WSSV vp28 Xu et al. (2007)

8. A case study of RNAi useA case study of RNAi use
• Place - Netherland
• Organism – Shrimp (P. monodon)
• Disease – WSD
• Causative agent – WSSV
• Gene construct – siRNA (vp28, vp15, gfp)
• Delivery – Intramuscular injection (40µl of 100µM
siRNA duplexes with buffer)
• Result – About 40 – 45% reduction in the mortality
where as control is having about 55 - 60%.
• Place - Netherland
• Organism – Shrimp (P. monodon)
• Disease – WSD
• Causative agent – WSSV
• Gene construct – siRNA (vp28, vp15, gfp)
• Delivery – Intramuscular injection (40µl of 100µM
siRNA duplexes with buffer)
• Result – About 40 – 45% reduction in the mortality
where as control is having about 55 - 60%.

9. APPLICATIONAPPLICATION
• Antiviral mechanism
• ‘Neo-females’ production
• Secures genome stability by keeping mobile
elements silent
• Offers a new experimental tool to repress genes
specifically
• GMO
• Investigate the functional role of specific genes
• Shelf life and bio-fortification
• Elimination of allergens and toxic substances
• Antiviral mechanism
• ‘Neo-females’ production
• Secures genome stability by keeping mobile
elements silent
• Offers a new experimental tool to repress genes
specifically
• GMO
• Investigate the functional role of specific genes
• Shelf life and bio-fortification
• Elimination of allergens and toxic substances

10. LIMITATIONSLIMITATIONS
• Most importantly effective delivery in vivo
• Off-target effects
• Triggering
• Innate immune responses
• Excessive levels of exogenous RNAs can saturate one
or more components of the endogenous RNAi
pathways, causing potential hazard
• Some viruses to escape RNAi mediated
• Most importantly effective delivery in vivo
• Off-target effects
• Triggering
• Innate immune responses
• Excessive levels of exogenous RNAs can saturate one
or more components of the endogenous RNAi
pathways, causing potential hazard
• Some viruses to escape RNAi mediated

11. CONCLUSIONCONCLUSION
• One of the most lucrative methodology of the
modern era but still has lot to understand in
terms of practical utility.
• Further study needs to be done in the field of
aquaculture practices.
• One of the most lucrative methodology of the
modern era but still has lot to understand in
terms of practical utility.
• Further study needs to be done in the field of
aquaculture practices.

12. •Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C.C., 1998. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391: 806–811.
•Kim, C. S., Kosuke, Z., Nam, Y. K., Kim, S. K. and Kim, K. H., 2007. Protection of shrimp (Penaeus chinensis) against white
spot syndrome virus (WSSV) challenge by double stranded RNA. Fish Shellfish Immunol., 23:242–246.
•Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S. and Rossi, J. J., 2004. Synthetic dsRNA Dicer substrates
enhance RNAi potency and efficacy. Nat. Biotechnol., 23: 222–226.
•Robalino, J., Browdy, C. L., Prior, S., Metz ,A., Parnel, P. and Warr, G., 2004. Induction of antiviral immunity by double
stranded RNA in a marine invertebrate. J Virol., 78: 10442– 10448.
•Wu, Y., Lu, L., Yang, L., Weng, S. and Chan, j., 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei
shrimp by sequence specific siRNA. Aquaculture, 271: 21–30.
•Westenberg, M., Heinhuis, B., Zuidema, D. and Vlak, J. M., 2005. siRNA injection induces sequence independent
protection in Penaeus monodon against white spot syndrome virus (WSSV). Virus Res., 114: 133–139.
•Xu, J., Han, F. and Zhang, X., 2007. Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA. Antiviral Res.,
73: 126–131.
•Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W. and Panyim, S., 2006. YHVprotease dsRNA inhibits YHV
replication in Penaeus monodon and prevents mortality. Biochem Biophys Res. Commun., 341: 351–356.
•Http://www.nature.com/nrg/rnai.html [Accessed on 9 December 2016]
•Fire, A., Xu, S. Q., Montgomery, M. K., Kostas, S. A., Driver, S. E. and Mello, C.C., 1998. Potent and specific genetic
interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391: 806–811.
•Kim, C. S., Kosuke, Z., Nam, Y. K., Kim, S. K. and Kim, K. H., 2007. Protection of shrimp (Penaeus chinensis) against white
spot syndrome virus (WSSV) challenge by double stranded RNA. Fish Shellfish Immunol., 23:242–246.
•Kim, D. H., Behlke, M. A., Rose, S. D., Chang, M. S., Choi, S. and Rossi, J. J., 2004. Synthetic dsRNA Dicer substrates
enhance RNAi potency and efficacy. Nat. Biotechnol., 23: 222–226.
•Robalino, J., Browdy, C. L., Prior, S., Metz ,A., Parnel, P. and Warr, G., 2004. Induction of antiviral immunity by double
stranded RNA in a marine invertebrate. J Virol., 78: 10442– 10448.
•Wu, Y., Lu, L., Yang, L., Weng, S. and Chan, j., 2007. Inhibition of white spot syndrome virus in Litopenaeus vannamei
shrimp by sequence specific siRNA. Aquaculture, 271: 21–30.
•Westenberg, M., Heinhuis, B., Zuidema, D. and Vlak, J. M., 2005. siRNA injection induces sequence independent
protection in Penaeus monodon against white spot syndrome virus (WSSV). Virus Res., 114: 133–139.
•Xu, J., Han, F. and Zhang, X., 2007. Silencing shrimp white spot syndrome virus (WSSV) genes by siRNA. Antiviral Res.,
73: 126–131.
•Yodmuang, S., Tirasophon, W., Roshorm, Y., Chinnirunvong, W. and Panyim, S., 2006. YHVprotease dsRNA inhibits YHV
replication in Penaeus monodon and prevents mortality. Biochem Biophys Res. Commun., 341: 351–356.
•Http://www.nature.com/nrg/rnai.html [Accessed on 9 December 2016]

14. Heros of CRISPR
George ChurchJennifer Doudna Feng ZhangEmmanuelle Charpentier
Thank you for attention
But big THANK YOU TO..