A simple version of the CRISPR/Cas system, CRISPR/Cas9, has been modified to edit genomes. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added.

1. CRISPR – Gene
Editing Technology
By – Romil J Mistry
7th Sem
M.Sc. Biotech.
Roll No. :10
Bhagwan Mahavir College of Science &
Technology, Surat

2. Discovery of CRISPR
M.Sc. BT 7th Sem.- BMCST, Vesu 2
YOSHIZUMI ISHINO
EMMANUELLE
CHARPENTIER
JENNIFER DOUDNA

3.  Clustered Regularly Interspaced Short Palindromic Repeats. CRISPR locus-
Nucleotide repeats (28-37 bp) with interspersed spacer DNA (32-38 bp) (Fig.1).
 Cas: A protein that cleaves foreign DNA.
 CRISPR and Cas proteins form CRISPR/Cas system which is a
prokaryotic immune system that confers resistance to foreign genetic elements such
as those present within plasmids and phages that provides a form of acquired
immunity. (Barrangou, et al., 2007). [1]
 Spacers are taken from viruses that previously attacked the organism which serve
as a bank of memories and enables bacteria to recognize the viruses and fight off
future attacks.
M.Sc. BT 7th Sem.- BMCST, Vesu 3

4. Future of CRISPR
M.Sc. BT 7th Sem.- BMCST, Vesu 4
• Could become a major force in ecology and conservation,
when paired with other molecular biology tools .
• Can be used to introduce genes that slowly kill off the
mosquitos spreading malaria .
• Introduce genes that put the brakes on invasive species like
weeds .
• Valuable tool in research .

5. Fig.2 Mechanism of CRISPR
MECHANISM
M.Sc. BT 7th Sem.- BMCST, Vesu 5

6. CRISPR/Cas systems-
1. Type I – recognises PAM sequence, require Cas3 and other Cas proteins for
interference, target the phage DNA.
2. Type II – recognises PAM sequence, requires solely Cas9 for interference, target
the phage DNA.
3. Type III – does not need PAM sequence, use Cas10 for interference, target the
phage mRNA.
 Protospacer Adjacent Motif /
PAM sequence
3-5 bp sequence (8-12 bp in humans)
found adjacent to the target DNA in the
phage genome that are recognised by
the Cas protein. (Ran, et al., 2013). [2]
Fig. 3 PAM Sequence
M.Sc. BT 7th Sem.- BMCST, Vesu 6

7. APPLICATIONS
1. GENOME ENGINEERING
a. Construction of Cas9 plasmid.
b. Transfection of the plasmid in the
host cell.
c. Expression of plasmid.
d. Activation of plasmid and its
binding to genomic target
sequence.
e. Cleavage of target DNA.
f. DNA repair by Non-homologous
End Joining (NHEJ) repair and
Homology directed repair (HDR).
M.Sc. BT 7th Sem.- BMCST, Vesu 7
Fig 4. CRISPR/Cas9 insertion in plasmid ( vector)

8. Fig.5 dsDNA break by Cas9
Source :
https://www.bsgct.org/crispr-cut-
paste-genome-level/
M.Sc. BT 7th Sem.- BMCST, Vesu 8

9. Fig.6 DNA repair by NHEJ and HDR
Source :
https://www.researchgate.net/figure/Do
uble-strand-breaks-repair-pathways-
During-homologous-recombination-
the-repair-of-DSB_fig2_257135774
M.Sc. BT 7th Sem.- BMCST, Vesu
9

10. Fig 7 Flowchart of the experimental design options for editing the genome using CRISPR-Cas9 [8]
M.Sc. BT 7th Sem.- BMCST, Vesu 10

11. 2. KNOCK-OUT / KNOCK-IN
o Knockdown- Repression
o Knock-in- Activation
• To study functional genomics and proteomics.
Fig. 8 Knock-in and Knock-out Fig. 9 CRISPR in Fish (Xie, et al., 2016)[7]
M.Sc. BT 7th Sem.- BMCST, Vesu 11

12. 3. GENE THERAPY
The resulting mice were fertile
and able to transmit the
corrected allele to their
progeny. The study provides
proof of principle for use of the
CRISPR-Cas9 system to
correct genetic disease. (Wu, et
al., 2013) [6]
4. CRISPR IN AGRICULTURE
o Adapted the CRISPR/Cas9
system for use in oleaginous
(oil-producing) yeast Yarrowia
lypolytica.
o Also for drought and disease
resistant wheat and rice.
Fig.10 CRISPR in disease modelling
M.Sc. BT 7th Sem.- BMCST, Vesu
12
Source
:https://www.sciencedirect.c
om/science/article/pii/S1934
590913004621

13. 5. DISEASE MODELLING
Marmosets are engineered that have a disrupted SHANK3 gene, is a candidate autism
gene (Shen, 2013)[4]. CRISPR-Cas9 was used to alter the DNA of the single-cell
monkey embryo causing the disrupted gene to be present in every cell of the primate.
This will better allow study of the gene’s role in autism and allow identification of
novel therapeutics.
Fig. 11 Marmosets are among the primates that may soon be engineered with custom genetic-editing
methods.
M.Sc. BT 7th Sem.- BMCST, Vesu
13
Source:
https://www.nature.com/news/prec
ision-gene-editing-paves-way-for-
transgenic-monkeys-1.14098

14. 6. BIOMEDICINE
o CRISPR is an effective way to limit replication of multiple herpesviruses.
o Able to eradicate viral DNA in the case of Epstein-Barr virus (EBV). Anti-
herpesvirus CRISPRs have promising applications such as removing cancer-
causing EBV from tumor cells, or preventing cold sore outbreaks and recurrent
eye infections by blocking HSV-1 reactivation. (van Diemen, et al., 2016) [5]
7. CRISPR IN CANCER
o On 21 June,2016, an advisory committee at the US National Institutes of Health
(NIH) approved a proposal to use CRISPR–Cas9 to help augment cancer
therapies that rely on enlisting a patient’s T cells, a type of immune cell.
o CRISPR would be used to alter T cells extracted from people with different
kinds of cancer and then administer those engineered T cells back to the same
people. (Reardon S, 2016) [3]
M.Sc. BT 7th Sem.- BMCST, Vesu 14

15. MERITS OF CRISPR
 High potency (cleavage efficiency and specificity)
 Broad applicability to both in vivo and ex vivo applications
 Simple editing tools (sgRNA and protein) allow unprecedented ability to
scale and optimize at speed
 Potential one-time curative treatment
 Ability to address any site in the genome or foreign genomes
 Ability to target multiple DNA sites simultaneously
 Multifunctional programmability: deletion, insertion or repair genes
DEMERITS OF CRISPR
 Off-target indels
 Limited choice of PAM sequences
 Ethical concerns regarding editing human embryos
M.Sc. BT 7th Sem.- BMCST, Vesu 15

16. 1. Barrangou, R., Fremaux, C., Deveau, H., Richards, M., Boyaval, P., Moineau, S., Horvath, P.
(2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science .
2. Ran, F. A., Hsu, P. D., Wright, J., Agarwala, V., Scott, D. A., Zhang, F. (2013). Genome
engineering using the CRISPR-Cas9 system. Nature protocols, 8(11).
3. Reardon, S. (2016). First CRISPR clinical trial gets green light from US panel. Nature, 531.
4. Shen, H. (2013). Precision gene editing paves way for transgenic monkeys. Nature, 503(7474), 14-
15.
5. van Diemen, F. R., Kruse, E. M., Hooykaas, M. J., Bruggeling, C. E., Schürch, A. C., van
Ham, P. M., Lebbink, R. J. (2016). CRISPR/Cas9-mediated genome editing of herpesviruses
limits productive and latent infections. PLoS pathogens, 12(6), e1005701.
6. Wu, Y., Liang, D., Wang, Y., Bai, M., Tang, W., Bao, S., Li, J. (2013). Correction of a genetic
disease in mouse via use of CRISPR-Cas9. Cell stem cell, 13(6), 659-662.
7. Xie, S. L., Bian, W. P., Wang, C., Junaid, M., Zou, J. X., and Pei, D. S. (2016). A novel
technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in
zebrafish. Scientific reports, 6, 34555.
8. Ethan Bier, Melissa M. Harrison, Kate M. O’Connor-Giles and Jill Wildonger , Advances in
Engineering the Fly Genome with the CRISPR-Cas System , GENETICS , January 1, 2018 ;vol.
208 no. 1 1-18
REFERENCES
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17. M.Sc. BT 7th Sem.- BMCST, Vesu 17