This presentation covered: Introduction; CRISPR Locus; Components of CRISPR; Cas9; Cas9 Lobes Guide RNA; Mechanism; Summary; Prons and Cons; Conclusion

1. Advance in Industrial Biotechnology Using
CRISPER Cas9 System
Group Members
Shahraiz Butt (Leader)(s2018231023)
M.Sufyan Nazeer(s2018231027)
Shagufta Arshad(s2018231061)
Rimsha Waheed(s2018231054)
Rahat Roshni(s2018231065)
School of Science, University of Management and Technology

2. Table of Contents
➢Introduction
➢CRISPR Locus
➢Components of CRISPR Cas9 System
➢About Cas9
➢Cas9 Lobes
➢Guide RNA
➢Mechanism
➢Summary
➢Prons and Cons
➢Conclusion

3. Introduction
➢CRISPR stands for clustered regularly interspaced short
palindromic repeats.
➢Genome editing technique
➢Remove, add and alter genome
➢Immune system in Bacteria and Archaea
➢Anti virus system
➢CRISPR locus is Vaccinated Card

4. CRISPR Locus
➢CRISPR locus consists of:
➢CRISPR Array, a short
sequence of nucleotide about
25-40
➢separated by short spacer
➢Cas gene encode protein like
endonucleases etc.

5. Components of CRISPR -Cas System
➢Spacer separates nucleotide sequence, drive from invading
viruses
➢Leader conserved sequence with CRISPR present upstream
➢Protospacer targeted sequence
➢PAM(protospacer associated motif) present next to target
sequence mecessary for recognition
➢crRNA is CRISPR RNA

6. About Cas9
➢Cas9 is RNA guided DNA endonuclease
➢Targets specific DNA sequence
➢crRNA require for Cas9 activation
Types of Cas9 are:
➢ Spy Cas9 (streptococcus pyrogene)
➢ StCas9 (streptococcus thermophilus)
➢ TdCas9 (Treponema denticola)

7. Cas9 Lobes
Cas9 protein has two lobes:
➢Recognition lobe
identification through PAM
➢Nuclease lobe- cleavage ,has
two domains:
➢HNH domain cleaves
complementary strand
➢RuvC domain cleaves non-
complementary strand

8. Guide RNA
➢Engineered 5’ end that is
complementary to target
DNA sequence.
➢Binds with Cas9, induces:
• Conformatinal changes
• Make it active

9. Mechanism
➢Cas9 searches for target sequence through PAM
➢Guide RNA has 5’ end complementary to target, helps in
binding
➢After binding HNH and RuvC will cut target DNA
➢DSB (double stranded break) will occur

11. Repairing of DSB
➢Blunt ends are repared by cell’s natural environment
Two mechanisms are followed:
➢NHEJ break ends directly ligated without the need of
homologous template
➢HDR require homologous template to guide repair

13. Summary

14. Pros and Cons
• PROS
➢Fast than others
➢Use in many species
➢Abilty to target any genomic
region
• CONS
➢off target effect
➢Ethical
➢Social effect in society

15. Conclusion
➢CRISPR has both advantages and disadvantages ranging
from ethical concern. BUT...!
This scientific breakthrough has the ability to:
➢Eliminate disease
➢To solve world hunger
➢Authorities sould formulate laws for its safe and ethical use