Crispr Cas 9 is a genome editing tool used in gene editing, this presentation explains the fundamental of how it works in a simple and easy manner.

1. Presented by:
Shruti Paliwal
En. no.: 190116014
M.Sc. (Ag) final year
Seminar Incharge:
Dr. A.K. Mehta
Dr. Yogendra Singh
Dr. Stuti Sharma
Major advisor:-
Dr. Stuti Sharma

3. Contents:-
 GENOME EDITING
 IMPORTANCE OF GENOME EDITING
 GENE EDITING TOOLS
 CRISPR TIMELINE
 WHAT IS CRISPR CAS9?
 KEY COMPONENTS OF CRISPR
 CRISPR AS A GENETIC TOOL
 CRISPR IN AGRICULTURE
 CASE STUDIES
 REFERENCE
 CONCLUSION

4. :GENOME EDITING:
 Genome editing, or genome editing with engineered nucleases
(GEEN) is a type of genetic engineering in which DNA is inserted,
replaced, or removed from a genome using artificially
engineered nucleases, or "molecular scissors”.
 The nucleases create specific double-strand breaks (DSBs) at
desired locations in the genome and harness the cell’s
endogenous mechanisms to repair the induced break by natural
processes of homologous recombination (HR) and non-
homologous end- joining (NHEJ).

5. Importance of 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. Fig: Process of genome
editing.
DNA strand is cut with
the help of nucleases,
which create a double
strand break in the
strand.
This double strand break
allow the possibilities of
inducing new change in
the genome.

7. GENE EDITING TOOLS
 Mega nucleases
 Zinc finger nucleases(ZFN)
 TALEN(Transcription activator like effector based
nucleases)
 Crispr cas9

8. Meganucleases
 Meganucleases (MNs) are encoded by mobile introns
sometimes known as transposons or selfish genetic elements.
 MNs asymmetric recognition sequence, which is usually 12–
40 bp long differentiates itself, from other Type-II restriction
enzymes, which have short (4–8 bp) recognition sequence.
 MNs occurs infrequently in the plant genome and therefore,
are specific to a given sequence, they represent, in most
cases.
 MNs works by using cell’s own repair mechanism and
therefore, bypassing proofreading mechanism to replicate
itself.
 Modulation of meganuclease target gene activity remains a
challenge and therefore, is less popular than other genome
editing techniques.

9. Zinc finger nucleases(ZFN)
 A ZFN is an enzyme with both a zinc finger DNA-binding domain
and also a restriction endonuclease domain.
 The first domain is used to target and bind to specific sequences
of DNA and the second domain is used to cleave the DNA at the
target site.
 The zinc-finger domain is made of a 3-base pair segment of DNA
that is designed to complement the target site and the
restriction endonuclease cleaves the site that it is guided to by
the first domain.
 ZFN was a huge breakthrough for site-specific gene editing, but
unfortunately it had some limitations.

10. Transcription activator-like effector nucleases
(TALENs)
 This method of gene editing is similar to ZFNs, being composed
of a DNA-binding domain and a second domain that cleaves the
DNA.
 TALENs are more useful than ZFNs because their DNA-binding
domains have more potential target sequences, meaning they
can be used for more applications.
 TALENs were a step forward from ZFNs, but they were expensive
to produce, despite being easier to design than ZFNs.
 There is another gene editing technique that uses restriction
enzymes along with recombinant adeno-associated viruses
(rAAVs).

11. Fig: working of nucleases
(a) ZFN: Zinc Finger
Nucleases .
(b)TALEN: Transcription
Activator-like Effector
nucleases.
(c) CRISPR/Cas9 :
clustered regularly
interspaced short
palindromic sequences.

14. Crispr Timeline:

25. What is Crispr cas9 ?
 CRISPR/Cas systems are part of the adaptive immune
system of bacteria and archaea, protecting them against
invading viruses by cleaving the foreign DNA in a
sequence-dependent manner.
 It has two components:
 1. The CRISPR loci: Composed of a series of repeats
interspaced by spacer sequences acquired from invading
genomes.
 2. Cas proteins: An RNA guided DNA endonuclease that
target and cleave invading DNA in a sequence specific
manner.

27. CRISPR Components:
 CRISPR has two components –
 a guide RNA (gRNA)
 CRISPR-associated endonuclease (Cas).
 The gRNA is specific to the target DNA sequence. In experiments
using CRISPR, the gRNA and Cas are combined and the result is a
ribonucleoprotein (RNP) complex.
 Cas is a protein that acts to cut the target DNA while the gRNA
guides it to the target DNA site. In prokaryotes, the gRNA is used
to target viral DNA, but as a gene editing tool, it can be designed
to target any gene site in almost any location.
 Cas9 is a popular choice of Cas proteins. Cas9 is generally taken
from Streptococcus spp.

28. Crispr Components:
 The gRNA finds the target site and binds to the DNA, but this
binding requires the presence of a PAM (protospacer adjacent
motif) immediately downstream of the target but on the
opposite DNA strand.
 Different Cas proteins (from different prokaryotic species)
recognise different PAM sequences. Cas9 is popular because of
the frequency and flexibility of its PAM, which is 5’-NGG-3’.
 The N represents any nucleotide. This means that any DNA
sequence with two G (guanine) bases can be used to form a PAM
for Cas9.
 If the gRNA successfully binds to the target, the Cas9 cleaves
both DNA strands. This cleaving process takes place 3 to 4
nucleotides upstream of the PAM site.

29. Guide RNA:
 In nature, gRNA is made of two separate sections of RNA.
These two sections are CRISPR RNA (crRNA) and
transactivating CRISPR RNA (tracrRNA). crRNA is 18-20 base
pairs in length and binds to the DNA target sequence.
 TracrRNA acts as a structure which the crRNA-Cas9
interaction can take place on. In nature, the gRNA is a duplex
molecule, with crRNA and tracrRNA annealed together.
 Synthetically, gRNA can be produced with these two
molecules connected by a linker loop. These are called single
guide RNAs (sgRNAs).

30. Fig: Components of
CRISPR Cas9
(a) Guide RNA
(b) PAM: Protospacer
Adjacent Motif
(c) tracrRNA:
transacctivating
crispr RNA
(d)crRNA: crispr RNA

35. Double Strand Break Repair
 CRISPR causes DSBs at desired sites in the genome. This is
just the first step in the editing process. It is the repair
mechanisms that follow this that allow the editing to
occur.
 Innate DNA repair processes automatically respond to
the formation of the DSB.
 The two main types of repair mechanisms that are used
to edit genes are :
 Non-homologous end joining (NHEJ)
 Homology-directed repair (HDR).

36. Non-Homologous End Joining(NHEJ)
 NHEJ can be used when the desired result is to Knock-In
Sequence permanently knockout a gene so that no
functional protein is made.
 NHEJ facilitates the re-joining of the DNA ends. However, it
often allows erroneous changes to occur, which may result
in inserted or deleted nucleotides that are not intended.
These are called indels.
 If the number of nucleotide changes (inserted or deleted)
are not a multiple of three, a frameshift mutation will occur.
This mutation will likely eliminate the functionality of the
resulting protein.

37. Homology Directed Repair(HDR)
 If the gene editing result desired by the researchers is to
replace the targeted DNA sequence with another
sequence, then HDR can be used.
 A DNA template from a donor that possessed that
desired DNA segment is introduced. This template is
surrounded by sections of homologous DNA sequences.
 The host’s repair mechanisms will use this template to fix
the DSB by using homologous recombination. By this
process, the donor’s sequence is incorporated into the
sequence being repaired.

38. Double Strand Break
(a) NHEJ: non
homologus end
joining
(b) HDR: Homology
Direct Repair

39. CRISPR function :
(a) Gene knockout
(b) Gene knock in

40. CRISPR interference (CRISPR i)
 In 2013, Qi et al. developed a variant of Cas9 that does not
cleave DNA. This was achieved by altering the endonuclease
domains through mutation. This new variant is called dead
Cas9 or dCas9.
 Using dCas9, a new technique was created where the dCas9
binds to the target DNA (but does not cleave it) and prevents
the host cell’s transcription machinery from reaching the
promoter.
 This prevents the gene from being expressed without cleaving
the DNA. Combining a transcriptional repressor domain with
dCas9 produces a mechanism that is reversible and effective
in inhibiting gene expression.

41. CRISPR interference (CRISPR i)
 The term CRISPR interference is a reference to its
precursor, the gene-silencing technique RNA interference
(RNAi). While RNAi destroyed RNA transcripts to silence
gene editing, CRISPRi affects genes by impacting DNA.
CRISPRi has higher efficiency, versatility and less
unintended (off-target) effects than RNAi.

42. Anti-CRISPR:
 CRISPR-Cas9 allows the fine-tuning of genomic DNA. However,
there is a downside. There is a risk of off-target effects, such
as cleaving the DNA in the wrong location.
 The solution is to use anti-CRISPR proteins that inhibit the
activity of Cas9. This can be seen in nature, when
bacteriophages use these types of proteins to deflect the
prokaryote’s CRISPR machinery.
 This technology can be applied to decrease errors in the
editing process.
 When the anti-CRISPR machinery is introduced after the
editing process occurs, the cleavage at on-target sites is only
partially decreased, while cleavage at off-target sites is greatly
decreased.

44. 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.

46. Proteinaceous body :
Gluten
Gluten is made up of
two protein called
gliadin and glutanin .

47. Gliadin gene in the
genome of wheat.

48. CRISPR Mutants:
1.Showed an 85%
decrease in
predicted
immunogenecity
2. Retain their
bread making
quality.
3.Retained
nutritional quality.
4.Showed reduced
immune response
in celliac and
gluten sensitive
patients.

50. (a) Fusarium
Head Blight , a
notorius wheat
pest.
(b)Deoxynivalenol
, or vomitotoxin.

51. Thinopyrum :
wheat grass , a
relatively
undomesticated
relative of wild
wheat.
It has got many
similarities with
wheat , their
leaves, flowering
time, pollination
time, seed shape
etc.
They can naturally
hybridize with
each other.

52. Here we isolated
the FHB resistance
gene Fhb7 by the
genome of
Thinopyrum
elongatum.
Fhb7 encodes a
glutathione S-
transferase (GST)
and confers broad
resistance to
Fusarium spp. by
detoxifying
trichothecenes
through
depoxidation.

54. Wild tomato
(Solanum
pimpenifolium)
Four genes were
mutanized which
resulted in the
modern day
cultivar(rich in
lycopene).

55. 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.

56. FUTURE PTOSPECTS:

57. Reference :
 RNA-guided genetic silencing systems in bacteria and archaea. Blake
Wiedenheft Samuel H. Sternberg & Jennifer A. Doudna. Nature 2012
 A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive
Bacterial Immunity. Jinek et al. Science 2012
 CRISPER handbook
 RNA-guided editing of bacterial genomes using CRISPR-Cas systems.
Jiang et al. Nature Biotech 2013
 CRISPR interference (CRISPRi) for sequence-specific control of gene
expression. Larson et al., Nature protocols 2013
 Targeted genome modifications in soybean with crispr/cas9. Jacobs et
al. BMC Biotechnology(2015)
 Large chromosomal deletions and heritable small genetic changes
induced by CRISPR/Cas9 in rice, Zhou et al. Nucleic acid research 2014