CRISPR technology- An emerging topic in translational research

1. CRISPR
-A new tool for genome editing
Presenter
Dr Amar Chandra Sharma
Post Doc Fellow (RA-ICMR)
Dept. of Orthopaedic Surgery,
KGMU

2. CRIPSR–Cassystems
These arethe part of bacterial immune systemwhich detectsand recognize the foreign
DNAand cleavesit.
1. TheCRISPR(Clustered Regularly Interspaced Short Palindromic Repeats) are DNA loci
containing short repetitions of base sequences which separatedby short "spacer DNA"
from previous exposures to a virus or phage.
2. Cas(CRISPR-associatedproteins) can target and cleave invading DNAin a sequence-
specific manner.
 It was first observed in Escherichia coli by Osaka University researcherYoshizumi Ishino in
1987.
 It represents a family of DNA repeats in most archaeal (~90%) andbacteria(~40%)
genomes provides acquired immunity against viruses and phages.

3. CRISPR
Spacer :- The direct repeats in a CRISP
.R locus are separated by short stretches of non-repetitive DNA called
spacers that aretypically derived from invading plasmid orphageDNA.
Protospacers :-The nucleotide sequence of the spacer must be similar to a region in the phage genome called a
protospacer in order to recognize and subsequently block phage replication, also known as
PAM(protospacer adjacent motif) seguence.
 The length and sequence of repeats and the length of spacers are well conserved within a CRISPR locus, but may
vary between CRISPRs in the same or differentgenomes.
 Repeat sequences are in the range of 21 bp to 48 bp, and spacers are between 26 bp and 72bp.
 A conserved sequence associated with CRISPR loci called leader, located up-stream of the CRISPR with respect to
direction of transcription.

4. The size of CRISPR repeats and spacers varies between 23 to 47
base pairs (bp) and 21 to 72 bp, respectively.
Generally, CRISPR repeat sequences are highly conserved within
a given CRISPR locus.
5

5. The spacer region of each CRISPR RNA base pairs with complementary
nucleic acids, driving cleavage or degradation by the Cas proteins (cas 1 and
Cas 2) within minutes of invasion.

6. History
 1987- CRISPR sequences were first discovered in Escherichia coli. (Ishino et al., 1987)
 2002- Identification of Cas genes that are associated with DNA repeats in prokaryotes.
(Jansen et al.,2002)
 2007- CRISPR provides acquired resistance against viruses in prokaryotes.
(Barrangou et al., 2007)
 A 2010 study provided direct evidence that CRISPR-Cas cuts both strands of
phage and plasmid DNA in S. thermophilus ( Garneau J.E, 2010. Nature. 468
(7320): 67–71)

7.  2012- Idea of using CRISPR- Cas9 as a genome engineering tool was published by
Jennifer Doudna and Emmanuelle Charpentier .
 Nobel prize in chemistry being awarded same in this year(2020)
Jennifer Doudna
Emmanuelle Charpentier
The discovery and characterization of the prokaryotic CRISPR-Cas immune system has
led to a revolution in genome editing and engineering technologies. Despite the fact that
most applications emerged after the discovery of the type II-A CRISPR-Cas9 system of
“Streptococcus pyogenes”.

8. Components of
CRISPR
1. Protospacer adjacent motif (PAM)
2. CRISPR-RNA (crRNA)
3. trans-activating crRNA (tracrRNA)
4. Cas proteins

9. Different Cas proteins and their function
Protei
n
Distributio
n
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 I
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 RAMP protein,

10. 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
expressio
n and
HD nuclease domain, palm domain, Zn
ribbon; some homologies with
CASCADE elements

11. 1
5
Variants of Cas9
Only one strand of DNA will be cut

12. Types of CRISPR CAS
system
 There arethree types of CRISPR-Cas systems,
which vary in their specific target and
mechanism of action.
 Type I systems cleave and degrade DNA,
 Type II systems cleave DNA ,
 Type III systems cleave DNA or RNA .

13. CRISPR-Cas Defense Mechanism
The CRISPR-Cas mediated defense
process can be divided into three
stages:
The first stage, adaptation, leads to
insertion of new spacers in the CRISPR
locus.
In the second stage, expression, the
system gets ready for action by
expressing the Cas genes and
transcribing the CRISPR into a long
precursor CRISPR RNA (pre-crRNA).
The pre-crRNA is subsequently
processed into mature crRNA by Cas
proteins and accessory factors.
In the third and last stage, interference,
target nucleic acid is recognized and
destroyed by the combined action of
crRNA and Cas proteins complex.

14. Guide RNA
Software for Guide RNA

15. Advantages of dCas9 modulation of gene expression (CRISPRi)
over RNAi technology
key distinctions between dCas9 and RNAi are that:
(a) dCas9 downregulation of transcripts acts at the DNA level, in
contrast to posttranscriptional regulation by RNAi,
expression,
that are not
(b) dCas9 offers the possibility of upregulating gene
allowing for discovery of gain-of-function phenotypes
possible to obtain with RNAi, and
(c) dCas9 can act on miRNAs and any other genomic region.

16. 45

17. Feluda: first Desi Covid detector
 An indigenously developed test that uses the cutting edge CRISPR gene-
editing technology to detect SARS-CoV-2 virus accurately, cheaply and
quickly.
 Feluda is the nickname of a fictional pvt detective in a series of popular
Bengali novels and films written and directed by “Mr. Satyajit Ray”.
 Developed by Scientists at council of scientific and industrial research’s
IGIB (Institute of Genomics and Integrative Biology) in Delhi with the
TATA group.

18. Comparision between sgRNA and crRNA-tracrRNA
hetero duplex
Advantages
• Flexible targeting
• Sequence specific
• Efficient
• Precise cleavage
• Affordable
• Quick
• Multiplex guides
Limitation
 Cas9 is a large protein
 PAM – dependent design limitations
 Off –target cleavage

19. Cas9-based tools are a formidable asset for studies in basic
science, biotechnology, and medicine. Cas9-based technologies
are spreading at a remarkable speed that speaks to their simplicity
and efficiency. One outstanding objective is the precise
determination of off-target effects and their consequences.
Although current data seem to indicate that these are not a
major obstacle, a better characterization of Cas9 cleavage is
required before the technology is used in humans.
Ethical considerations are a second outstanding issue
Conclusion