This document provides an overview of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and its role as an adaptive immune system in prokaryotes. It describes the components and function of the CRISPR-Cas system, including how it provides immunity against viruses and plasmids. Applications of CRISPR technology discussed include phage resistance in bacteria, gene regulation, and bacterial strain typing. Potential future uses involve harnessing CRISPR biology for applications like transcriptional control.

1. CRISPR - A New Tool inCRISPR - A New Tool in
Molecular BiologyMolecular Biology
Presented by-
Musharraf Ali
M.Tech- 2 sem
Roll No.-15012006
School of BCE , IIT-BHU

2. CRISPR-
• Clustered regularly interspaced short palindromic repeats
• found in PK DNA as repeats followed by a short spacer
DNA segment
• spacer DNA segment could contain previously exposured
bacterial plasmid or virus
• contributed as an adaptive/acquired immunity in PK
system.
• common in archae bacteria

3. Kind of Repeats observed-
• Regularly Spaced Repeats
• Direct Repeats in Mycobacteria
• TREPs in Haloarchaea
• short repeat (SR) arrays
Later, Jansen and Mojica proposed replacing SRSR with the
more explicit, elegant and now widely adopted acronym
CRISPR, for Clustered Regularly Interspaced Short
Palindromic Repeats .

4. Distribution of CRISPR Arrays Amongst
Archaea and Bacteria

5. CRISPR-Cas Function Revealed
• Early Hypotheses-
- modulating gene expression
- binding sites for regulatory proteins
- regulation of neighbouring genes
- role in recombination
- role in replicon partitioning
- chromosomal rearrangement

6. • The Link to Invading Genetic Elements
- Two reports in 2005 revealed the presence of spacer
- analyses of isolates of Streptococcus pyogenes and
Yersina pestis
- BLAST searches s revealed that 2 % of
analysed spacers showed close similarities to viral DNA or
plasmid DNA
- CRISPR-Cas system in immunity was demonstratedCRISPR-Cas system in immunity was demonstrated
experimentally forexperimentally for S.thermophilus in 2007

7. Functional Components of CRISPR-Based
Systems
• composed of -
- regularly alternating repeats and spacers
- a leader sequence at one end of the array
- a set of cas genes
these components participate in different stages
of the CRISPR-Cas pathway, generating CRISPR-RNAs
(crRNAs) and Cas proteins

8. Fig- Schematic representation of CRISPR-Cas systems and the main steps of
CRISPRmediated interference

9. Identification of CRISPR-associated
Proteins
• Four cas (CRISPR-associated) genes, cas1–cas4, were originally
identified
• Cas1 and Cas2, the Cas3 and Cas4 protein families were
proposed to be homologous to DNA-helicases and
exonucleases, respectively
• CRISPR-Cas systems into three main types (I, II and III) on
the presence of particular signature cas genes

11. APPLICATION
1- Resistance Against Viruses
Dairy fermentations using Streptococcus thermophilus.
3 approaches are-
a)-‘‘CRISPerization’’: Phage Resistance Improvement
b)-Artificial Spacer Engineering
c)-Transfer Between Microorganisms

12. 2- Immunity Against Non-Viral Nucleic Acids-
-provides immunity against nucleic acids through base pairing between
spacer-derived crRNAs and complementary target sequences
Beside immunity it also confer-
a)-Plasmid Interference-
In Staphylococcus epidermidis where CRISPR-encoded spacers lowered
efficiency of plasmid uptake.
b)-Interference Against Other Mobile Elements –
CRISPR-mediated interference against antibiotic resistance genes.
ability of S. thermophilus to naturally acquire spacers that target an
antibiotic resistance gene

13. 3- CRISPR-Based Gene Regulation –
•potential to use CRISPR-Cas systems for the regulation, transcriptional
control, or regulation of transcript levels within a cell (patent
application WO/2010/075424)
•ability of CRISPR spacers to lower transcript levels, showing that a
spacer homologous to the histidyl-tRNA synthetase sequence lowers
His-tRNA levels (Aklujkar and Lovley 2010)
•Analogies between CRISPR-mediated interference and RNA
interference
•to harness the flexibility and modularity of CRISPR-Cas systems for RNA
interference in bacteria and archaea(patent applications WO/
2010/011961 and WO/2010/054108)

15. 4- CRISPR-Based Strain Typing –
-CRISPR loci comprise of two main elements, the CRISPR spacer array
and a group of CRISPR-associated (cas) genes
-CRISPR applications existed long before their function was elucidated.
The first use of spacer information for subtyping was in spacer-
oligonucleotide typing, or “spoligotyping,” of Mycobacterium
tuberculosis strains.
-principle of spoligotyping is PCR amplification of the CRISPR array with
labeled primers that recognize the DR sequences

17. Patent applications related to various uses of
CRISPR-Cas systems
Publication number Title Inventor Publication Date
WO/2006/073445 Detection and typing
of bacterial strains
Russell et al. 13.07.2006
WO/2007/025097 Use of CRISPR-
associated genes (cas)
Horvath et al. 01.03.2007
WO/2007/136815 Tagged
microorganisms and
methods of tagging
Barrangou et al. 29.11.2007
WO/2008/108989 Cultures with
improved phage
resistance
Barrangou et al. 12.09.2008

18. CONCLUSION & FUTURE PERSPECTIVE
• CRISPR-based typing techniques have been well established
for some bacterial species such as Mycobacterium and are
currently being developed and studied extensively,
especially for human pathogens but also for some
agriculturally important species
• strain-specific portions of a CRISPR allele using spacer-
specific primers or third-generation long-read sequencing
• Harnessing CRISPR biology, specifically, identifying the
presence/ absence of spacer sequences for subtyping
purposes

19. REFRENCES
• Deveau, H., Garneau, J. E. & Moineau, S. CRISPR/Cas system and its
role in phage-bacteria interactions. Annu. Rev. Microbio.
• Horvath, P. & Barrangou, R. CRISPR/Cas, the immune system of
bacteria and archaea. Science.
• Mojica, F. J., Diez-Villasenor, C., Soria, E. & Juez, G. Biological
significance of a family of regularly spaced repeats in the genomes of
Archaea, Bacteria and mitochondria. Mol. Microbiol.
• Marraffini, L. A. & Sontheimer, E. J. CRISPR interference limits
horizontal gene transfer in staphylococci by targeting DNA. Science
• Garneau, J. E. et al. The CRISPR/Cas bacterial immune system cleaves
bacteriophage and plasmid DNA. Nature
• Konermann, S. et al. Genome-scale transcriptional activation by an
engineered CRISPR–Cas9 complex. Nature

20. FURTHER INFORMATION
>Eugene V. Koonin’s homepage:
http://www.ncbi.nlm.nih.gov/CBBresearch/Koonin/ COGs:
http://www.ncbi.nlm.nih.gov/COG/grace/generin.cgi
>CRISPRdb: http://crispr.u-psud.fr/crispr/
>NCBI CRISPR/Cas: ftp://ftp.ncbi.nih.gov/pub/wolf/_suppl/
CRISPRclass/index.html
>PDB: http://www.rcsb.org/pdb/home/home.do TIGRFAMs:
http://www.jcvi.org/cgi-bin/tigrfams/index.cgi
>CRISPRi protocol by Nature Protocols: http://www.nature.
com/nprot/journal/v8/n11/full/nprot.2013.132.html