CRISPR-CAS System: From Adaptive Immunity To Genome editing. CRISPR Biology,

1. Presented by : Arpan Mukherjee (PhD scholar of IISER
Bhopal) and Debanjan Pandit Faculty Of Raidighi College

2. CRISPR System: From
Adaptive Immunity to
Genome Editing
---JENNIFER DOUDNA

4. CRISPRs : Clusters of
Regularly Interspaced Short
Palindromic Repeats
And what is a
CRISPR???????
It really refers to a pattern of DNA
sequences that are found naturally in
many bacterial genomes and other kinds
of single-celled organisms as well that
include a series of DNA
sequences that are repeated again and
again.
These black diamonds often have partial
palindromic characters so they can be
read in the forward and reverse direction
in the same way. So they have the
possibility, if you look at the letters of
DNA, they have the possibility of base
pairing to form a cruciform sort of
structure.

5. Functional CRISPR-Cas loci comprise a CRISPR array ofidentical repeats intercalated with invader
DNA-targeting spacers that encode the crRNA components and an operon of cas genes encoding the Cas
protein components.

6. Adaptive immunity occurs in three stages: (Mode of Action):
(i)Insertion of a short sequence of the invading DNA as a spacer sequence into the CRISPR
array
(ii)Transcription of precursor crRNA (pre-crRNA) that undergoes maturation to generate
individual crRNAs, each
composed of a repeat portion and an invader targeting spacer portion
(iii) crRNA-directed cleavage of foreign nucleic acid by Cas proteins at sites
complementary to the crRNA spacer sequence
The protospacer adjacent motif (PAM), a short sequence motif adjacent to the crRNA-targeted
sequence on the in-vading DNA, plays an essential role in the stages of adaptation and interference
in type I and typeII systems.

8. Mechanism
of CRISPR
Cas as
Adaptive
immunity

9. Applications of CRISPR-Cas System
1. CRISPR/Cas system is particularly beneficial in plant breeding because multiple traits can be
modified simultaneously.
2. One further application of CRISPR/Cas9 that is likely to expand in the future is the targeted
insertion of transgenes in the fields of metabolic engineering and molecular farming, where plants
or plant cells are used as factories for the production of specific metabolites or proteins.

10. Applications beyond Genome editing:
1. Gene regulation
A catalytically inactive dead Cas9(dCas9) can be fused to either a transcriptional
repressor or activator. When the dCas9-repressor fusion is recruited by a
gRNA(guide RNA) that matches the promoter, 5’untranslated region or coding
sequence of an endogenous gene, it can block transcription initiation, elongation or
the binding of transcription factors.
When the dCas9-activator fusion is targeted to a promoter, the specific expression
of the endogenous gene is induced.

11. 2.Cargo delivery. The catalytically inactive dCas9 can also be exploited as a programmable
DNA-binding protein to deliver diverse cargos to specific genomic locations.
For example, fusion with a green fluorescent protein(GFP)(left) provides a tool for visualizing
chromosome structure or dynamics, and fusion with a demethylase (right) can be used for targeted
epigenome editing.

12. RNA cleavage.
The Type III-B CRISPR-Cas system (e.g., from Pyrococcus furiosus) is composed of
nucleases that form the so-called Cmr9(Cas protein) complex (yellow) and represents a
unique RNA silencing system. The Cmr-crRNA complex targets invading RNA in a PAM-
independent process and is able to degrade complementary RNA sequences cleaving them
at multiple sites.

13. --THANK YOU--