Introduction to Crispr-cas system mediated defense system, its stages, types and applications

1. CRISPR-cas System
Presenter
Hajra
The Women University Multan

2. Table of Contents
• Introduction to CRISPR-cas system
• Stages of CRISPR-cas system mediated
defense
• Types of CRISPR-cas system mediated
defense
• Applications

3. CRISPR-cas system
• An array of short repeated sequences separated by spacers in a region of
DNA called Clustered Regularly Interspaced Short Palindromic Repeats
(CRISPR)
• The spacers derived from nucleic acid of viruses and plasmids and act as
recognition elements to find matching virus genomes
• Antiviral defense system
• Targets DNA or RNA protecting against viruses and other mobile genetic
elements

4. • The CRISPR locus,first observed in Escherichia coli
• Found on both chromosomal and plasmid DNA
• CRISPR activity requires the presence of a set of CRISPR-
associated (cas) genes
• Code for proteins essential to the immune response
• Offspring inherit the protection due to genome modification by
spacer acquisition

5. Stages of CRISPR-cas
mediated defense

6. • The key steps of CRISPR-cas immunity.
• 1) Adaptation: insertion of new spacers into the CRISPR locus.
• 2) Expression: transcription of the CRISPR locus into long
precursor CRISPR RNA and processing of pre-CRISPR RNA
into mature crRNA by cas proteins and accessory factors
• 3) Interference: detection and degradation of mobile genetic
elements by CRISPR RNA and cas protein

7. Stages of CRISPR-cas mediated defense

8. Types of CRISPR-cas
systems

9. Type I CRISPR-cas system
• Signature protein Cas3, a protein with both helicase and DNase domains
responsible for degrading the target
• Cascade binds crRNA and locates the target, and most variants are also
responsible for processing the crRNA
• Complex of different Cas proteins---Cascade (CRISPR-associated
complex for antiviral defense)

10. • The first 6–12 nt of the crRNA spacer are most important for target
binding and are termed the seed sequence
• Production of R-loop by base pairing of the crRNA with the
complementary DNA strand and additional displacement of the non-
complementary strand
• Cascade recruits Cas3 to degrade the targeted viral DNA molecule
• Dissociation of Cascade and ready for action again
• No base pairing of 5' terminal tag of the crRNA and the PAM sequence to
avoid host genomic encoded CRISPR cluster degradation

12. Type II CRISPR-cas system
• Require only the Cas9 protein for adaptation, crRNA expression
and interference
• in addition tracrRNA and the crRNA
• trans-activating crRNA (tracrRNA) contains a 25 nt long stretch
that is complementary to the crRNA repeat sequence
• Two separate lobes for target recognition and nuclease activity

13. • Recognition lobe is important for binding crRNA and target DNA
• The nuclease lobe cleave the complementary and non-complementary
strands of the target
• Cas9 ---RNA duplex formation by base pairing of tracrRNA and pre-
crRNA
• Cleavage of duplex by RNase III generates mature crRNAs
• In the interference Cas9 cleaves the DNA strand complementary to the
crRNA and non complementary strand

15. Type III CRISPR-cas system
• pre-crRNA maturation by sequence-specific processing step mediated by Cas6
• Yield mature crRNAs with a defined 5' end and variable 3' end
• Interference by complex of Csm or Cmr proteins
• Csm complexes target DNA and Cmr complexes targets RNA
• No PAMs are detected for Type III systems
• Extension of crRNA base pairing into the repeat region of host DNA---self-
nonself descrimination

17. • Only the type II CRISPR/Cas systems are established for genome editing
or gene silencing
• Difference between the three major CRISPR/Cas types
• The interference reaction of both, type I and III systems, relies on multi-
protein complexes
• Protein-engineering of a single Cas9 protein is more straightforward than
optimization of Cascade or Cmr/Csm complexes
• No need of PAM sequences for interference should be advantageous for
more versatile editing events

18. Applications

19. • In genotyping method is known as spacer oligonucleotide typing
(spoligotyping) to identify closely related bacterial strains e.g. M.
tuberculosis
• In dairy industry, production of Danisco----marketing starter cultures
for improved cheese production and other applications. The cultures
contain bacteria that have CRISPRs with improved resistance to
phages
• Cas9 produces a single double-stranded break in the DNA, an
important feature of a gene-editing tool

20. • Cas9 has been developed as an antimicrobial agent that can be used
to specifically target antibiotic-resistant and/or highly virulent
strains of bacteria
• Gene therapy applications have also been demonstrated by repairing
the cftr gene in cultured cells from human cystic fibrosis patients
• Cas9 also holds potential for treatment of viral infections, as
demonstrated for HIV and hepatitis B

21. • CRISPR-Cas systems used for gene silencing by interfering
with RNA polymerase binding or elongation
• A short version of Cas9 to be deliverable by Adeno-associated
virus, greatly facilitating its use in somatic gene therapy