Gene Silencing, Methods and emphasis on CRISPR Technology, CRISPR-CAS Technology.

1. Genetic Engineering
(BTMT-502)
Submitted By:-
Ilika Kaushik
19PBT004
M.Tech Biotechnology,
2nd Sem,
SoBT, GBU.
Submitted To:-
Dr. Bhaswati Banerjee
Ma’am
Asst. Professor
SoBt, GBU

2. Topic
New Frontiers in Gene Silencing and Genome:-
A Timeline of Technologies with special
emphasis on CRISPR-CAS

3. Introduction
• Gene Silencing- Regulation of gene expression in a
cell, to prevent the expression of a certain gene.
• Genome- In the fields of molecular biology and
genetics, a genome is the genetic material of an
organism. It consists of DNA. The genome includes
both the genes and the noncoding DNA, as well as
mitochondrial DNA and chloroplast DNA. The
study of the genome is called genomics.

4. • Gene Silencing can occur during transcription or
translation.
• Often used in research.
• Methods used to silence genes are now being used
to produce therapeutics.
• Gene silencing is often considered as gene
knockdown.
• When genes are silenced, their expression is
reduced.
• Gene silencing is referred to as gene knockdown
because the expression is reduced generally by 70%.

5. Why Genome Editing?
• To understand the function of a gene or a protein, one
interferes with it in a sequence-specific way and
monitors its effects on the organism.
• In some organisms, it is difficult or impossible to
perform site-specific mutagenesis, and therefore more
indirect methods must be used, such as silencing the
gene of interest by short RNA interference (siRNA).
• But sometime gene disruption by siRNA can be
variable or incomplete.
• Nucleases such as CRISPR can cut any targeted
position in the genome and introduce a modification of
the endogenous sequences for genes that are
impossible to specifically target using conventional
RNAi.

6. Types
Transcriptional Post-Transcriptional Meiotic
Genome Imprinting RNA Interference Transvection
Paramutation RNA Silencing Meiotic Silencing of
Unpaired DNA
Transposon Silencing
(Histone Modification)
Nonsense Mediated Decay
Transgene Silencing
Position Effect
RNA directed DNA
Methylation

7. Methods of Gene Silencing

8. 1.Antisense Oligonucleotides
• Discovered in 1978 by Paul Zamecnik and Mary
Stephenson.
• Oligonucleotides are short nucleic acid fragments,
bind to complementary target mRNA molecules
when added to the cell.
• Single-Stranded DNA or RNA, 13-25 nucleotides
long.

9. The antisense oligonucleotides can
affect gene expression in two ways:
RNAse H-dependent mechanism
• Target the mRNA molecules
to be degraded.
• More efficient.
• 80%-90% decrease in
protein and mRNA
expression.
Using a steric blocking
mechanism
• Prevent translation of
mRNA molecule.

10. RNAse H-dependent mechanism

11. 2.Ribozymes
• Ribozymes are catalytic molecules used to inhibit
gene expression.
• These molecules work by
cleaving mRNA molecules, essentially silencing the
genes that produced them.
• Sidney Altman and Thomas Cech first discovered
catalytic RNA molecules, RNase P and group II
intron ribozymes, in 1989 and won the Nobel Prize
for their discovery

12. • The general catalytic mechanism used by ribozymes is
similar to the mechanism used by
protein ribonucleases.
• These catalytic RNA molecules bind to a specific site
and attack the neighbouring phosphate in the RNA
backbone with their 2' oxygen, which acts as
a nucleophile, resulting in the formation of cleaved
products with a 2'3'-cyclic phosphate and a 5' hydroxyl
terminal end.
• This catalytic mechanism has been increasingly used by
scientists to perform sequence-specific cleavage of
target mRNA molecules.
• In addition, attempts are being made to use ribozymes
to produce gene silencing therapeutics, which would
silence genes that are responsible for causing diseases.

14. 3.RNA Interference
• RNA interference (RNAi) is a natural process used by cells to
regulate gene expression. It was discovered in 1998
by Andrew Fire and Craig Mello, who won the Nobel Prize
for their discovery in 2006.
• The process to silence genes first begins with the entrance of
a double-stranded RNA (dsRNA) molecule into the cell,
which triggers the RNAi pathway. The double-stranded
molecule is then cut into small double-stranded fragments by
an enzyme called Dicer.
• These small fragments, which include small interfering RNAs
(siRNA) and microRNA (miRNA), are approximately 21–23
nucleotides in length. The fragments integrate into a multi-
subunit protein called the RNA-induced silencing complex,
which contains Argonaute proteins that are essential
components of the RNAi pathway.

15. • One strand of the molecule, called the "guide"
strand, binds to RISC, while the other strand, known
as the "passenger" strand is degraded.
• The guide or antisense strand of the fragment that
remains bound to RISC directs the sequence-specific
silencing of the target mRNA molecule. The genes
can be silenced by siRNA molecules that cause the
endonucleatic cleavage of the target mRNA
molecules or by miRNA molecules that suppress
translation of the mRNA molecule.
• With the cleavage or translational repression of the
mRNA molecules, the genes that form them are
rendered essentially inactive.

17. 4.Three Prime Untranslated Regions and
MicroRNAs
• Three prime untranslated regions (3'UTRs) of messenger
RNAs (mRNAs) often contain regulatory sequences that post-
transcriptionally cause gene silencing.
• Such 3'-UTRs often contain both binding sites for microRNAs
(miRNAs) as well as for regulatory proteins. By binding to
specific sites within the 3'-UTR, a large number of specific
miRNAs decrease gene expression of their particular target
mRNAs by either inhibiting translation or directly causing
degradation of the transcript, using a mechanism similar to
RNA interference.
• The 3'-UTR also may have silencer regions that bind repressor
proteins that inhibit the expression of an mRNA.

18. • The 3'-UTR often contains microRNA response
elements (MREs).
• MREs are sequences to which miRNAs bind and
cause gene silencing.
• These are prevalent motifs within 3'-UTRs. Among
all regulatory motifs within the 3'-UTRs (e.g.
including silencer regions), MREs make up about
half of the motifs.

20. Comparison between Traditional and
Modern Genome Editing Technologies
Mutagen Chemical(e.g.,
EMS)
Physical (e.g.,
gamma, X- ray
or fast neutron
radiation)
Biological
(ZFNs, TALENs
or CRISPR/
Cas)
Biological-
Transgenics
(e.g., Agro or
gene gun)
Characteristics
of genetic
variation
Substitution and
Deletion
Deletion and
chromosomal
mutation
Substitution and
Deletion and
insertion
Insertions
Loss of function Loss of function Loss of function and
gain
of function
Loss of function and
gain
of function
Advantages Unnecessary
of knowing
gene function
or sequences
Unnecessary of
knowing gene
function or
sequences
Gene specific
mutation
Insertion of genes
of known functions
into host plant
genome
Easy production of
random
mutation
Easy production of
random mutation
Efficient production
of
desirable mutation
Efficient creation of
plants
with desirable traits

21. Mutagen Chemical(e.g.,
EMS)
Physical
(e.g.,
gamma, X-
ray or fast
neutron
radiation)
Biological (ZFNs,
TALENs or
CRISPR/ Cas)
Biological-
Transgenics (e.g.,
Agro or gene gun)
Disadvantages Inefficient
screening of
desirable traits
Inefficient
screening
of desirable
traits
Necessity of
knowing gene
function and
sequences
Necessity of
knowing gene
function and
sequences
Non specific
mutation
Non specific
mutation
Prerequisite
of efficient
genetic
transformati
on
Prerequisite of
efficient
genetic
transformation
Other features Non transgenic
process
and traits
Non
transgenic
process and
traits
Transgenic
process but non
transgenic traits
Transgenic process
and traits

22. 1987
• Researchers find CRISPR sequences in Escherichia coli, but do not characterize
their function.
2000
• CRISPR sequence are found to be common in other
microbes.
2002
• Coined CRISPR name, defined signature Cas genes.
2007
• First experimental evidence for CRISPR adaptive immunity.
2013
• First demonstration of Cas9 genome engineering in eukaryotic cell.

23. CRISPR – Cas Systems
• These are thepart of the Bacterial immune system
which detects and recognize the foreign DNA and cleaves it.
THE CRISPR (Clustered Regularly Interspaced Short Palindromic
Repeats) loci
Cas (CRISPR- associated) proteins can target and cleave invading DNA
in a sequence – specific manner.
• ACRISPRarray is composed of a series of repeats
interspaced by spacer sequences acquired from
invading genomes.

24. Components
of CRISPR
Protospacer
adjacent
motif (PAM)
Trans-
activating
crRNA
(tracrRNA)
CRISPR-
RNA
(crRNA)

25. Different CRISPR-Cas system in Bacterial Adaptive
Immunity
Class 1- type I (CRISPR-Cas3) and
type III (CRISPR- Cas10)
 uses several Cas proteins and the
crRNA
Class 2- type II (CRISPR-Cas9)
and type V (CRISPR- Cpf1)
 employ a large single-component
Cas-9 protein in conjunction with
crRNA and tracerRNA.
Zetsche et al.,
(2015)
functioning of type II
CRISPR system

26. Different Cas Proteins and their Function
Protein Distribution Process Function
Cas1 Universal Spacer Acquisition DNAse, not sequence specfic, can bind RNA;
present in all types.
Cas2 Universal Spacer Acquisition Specific to U-rich regions; present in all types.
Cas3 Type II Signature Target Interference DNA Helicase, Endonuclease
Cas4 Type I,II Spacer Acquisition RecB-like nuclease with exonuclease activity
homologous to RecB.
Cas5 Type I crRNA Expression RAMP protein, endoribonuclease involved in
crRNA biogenesis; part of CASCADE.
Cas6 Type I, III crRNA Expression RAMP protein, endoribonuclease involved in
crRNA biogenesis; part of CASCADE.
Cas7 Type I crRNA Expression RAMP protein, endoribonuclease involved in
crRNA biogenesis; part of CASCADE.
Cas8 Type I crRNA Expression Large protein with McrA/HNH-nuclease domain
and RuvC-like nuclease; part of CASCADE.
Cas9 Type II Signature Target Interference Large multidomain protein with McrA-HNH
nuclease domain and RuvC-like nuclease domain;
necessary for interference and target cleavage.
Cas10 Type III Signature crRNA Expression
and Interference
HD nuclease domain, palm domain, Zn ribbon;
some homologies with CASCADE elements.

27. Action of CRISPR in Bacteria.
• The CRISPR immune system works to protect bacteria from
repeated viral attack via three basic steps:

29. Structure of Cas9 Protein

30. Structure of crRNA

34. Versatile Nature of CRISPR Technology
(Jeffry et al., 2014)

35. Combining crRNA and tracrRNAinto sgRNA was the crucial step
for the development of CRISPR Technology
(Joung et al., 2012)

36. What makes CRISPR system the ideal
genome engineering technology?
Key enabling attributes to
become next big drug class.
• High potency (cleavage
efficiency) and specificity.
• Broad applicability to both
in vivo and ex vivo
applications.
• Simple editing tools allow
unprecedented ability to
scale and optimize at speed.
• Potential one time curative
treatment.
Broadest potential to modulate
genes.
• Ability to address any site in
the genome or foreign
genomes.
• Ability to target multiple
DNA sites simultaneously.
• Multifunctional
programmability:- delete,
insert or repair genes.

37. Examples of crops modified with CRISPR Technology
CROPS DESCRIPTION REFERNCES
Corn Targeted mutagenesis Liang et al. 2014
Rice Targeted mutagenesis Belhaj et al. 2013
Sorghum Targeted gene modification Jiang et al. 2013b
Sweet orange Targeted genome editing Jia and Wang 2014
Tobacco Targeted mutagenesis Belhaj et al. 2013
Wheat Targeted mutagenesis Upadhyay et al. 2013, Yanpenget
al. 2014
Potato
Soybean
Targeted mutagenesis
Gene editing
Shaohui et al., 2015
Yupeng et al., 2015

38. Genome editing
Tool
Transformation
Method
Crops Targeted genes

39. General Protocol for CRISPR

41. Recent Advances
Discovery of
new version of
Cas9.
Engineered Cas9
with altered PAM
specificity

42. • Cpf1 (CRISPR from Prevotella and Francisella
1) at Broad Institute of MIT and Harvard,
Cambridge.
• CRISPR-Cpf1 is a class 2 CRISPR system
• Cpf1 is a CRISPR-associated two-component
RNA programmable DNA nuclease
• Does not require tracerRNA and the gene is 1kb
smaller
• Targeted DNA is cleaved as a 5 nt staggered cut
distal to a 5’ T-rich PAM
• Cpf1 exhibit robust nuclease activity in human
cells Zetsche et al., (October
22, 2015)
New Version of Cas9

43. Cpf1 makes staggered
cut at 5’ distal end
from the PAM
Organization of two CRISPR loci found in
Francisella novicida .The domain
architectures of FnCas9 and FnCpf1 are
compared

44. DNAi-Targeted DNA
Degradation
Brian J. et al.,
2015
• Once an engineered organism completes its task, it is useful to degrade the
associated DNA toreduce environmental release and protect intellectual
property.
• Here is a genetically encoded device (DNAi) that responds to a
transcriptional input and degradesuser- defined DNA.
• This enables engineered regions to be obscured when the cell enters a new
environment.
• DNAi is based on type-IE CRISPR biochemistry and a synthetic CRISPR
array defines the DNAtarget.
• When the genome is targeted, this causes cell death, reducing viable cells by
a factor of10^8

45. Application in Agriculture
• Can be used to create high degree of genetic variability at precise
locus in the genome of the crop plants.
• Potential tool for multiplexed reverse and forward genetic study.
• Precise transgene integration at specific loci.
• Developing biotic and abiotic resistant traits in crop plants.
• Potential tool for developing virus resistant crop varieties.
• Can be used to eradicate unwanted species like herbicide
resistant weeds, insect pest.
• Potential tool for improving polyploid crops like potato and wheat.

46. Some pitfalls of this
Technology
• Proper selection of gRNA
• Use dCas9 version of Cas9 protein
• Make sure that there is no mismatch
within the seed sequences(first 12 nt
adjacent to PAM)
• Use smaller gRNA of 17 nt instead of 20
nt
• Sequence the organism first you want
to work with
• Use NHEJ inhibitor in order to boost
up HDR
3
3
Solutions
• Off target indels
• Limited choice of PAM
sequences.

47. How to avoid off-target
effects?
• Optimization of Injection conditions (less
cas9/sgRNA)
• Bioinformatics:- Find a sgRNA target for
less off-targets “CRISPR Design”
(http://crispr.mit.edu)

48. Conclusion…
• Genome editing tools provide new strategies for genetic manipulation in
plants and are likely to assist in engineering desired plant traits by modifying
endogenous genes.
• Genome editing technology will have a major impact in applied crop
improvement and commercial product development .
• CRISPR will no doubt be revolutionized by virtue of being able to
make targeted DNA sequence modifications rather than random changes.
• In gene modification, these targetable nucleases have potential applications
to become alternatives to standard breeding methods to identify novel traits
in economically important plants and more valuable in biotechnology as
modifying specific site rather than whole gene.

50. s