Terminologies Repeats Spacer CAS protein CRISPR RNA (crRNA) Trans activating crRNA ( tracrRNA Guide RNA (gRNA) Protospacer adjacent motifs (PAMs) CRISPR System •CRISPR locus ( Clustered Regularly Interspaced Short Palindromic Repeats) •Natural immune system of bacteria and archaea, similar to RNAi mechanism in eukaryotes Tools for Genetic Engineering DNA structure/Sequencing Restriction enzymes PCR Genome editing(CRISPR/CAS) Applications of CRISPR Agriculture : USDA approved CRISPR edited food as safe as ones bred from conventional way Gene Therapy & Medicine: Treatment of genetic disease, Cancer, blindness, Alzheimer’s disease, HIV, etc. Biofuel and Chemicals: increased oil yield from GE strain of ocean algae Transgenic Animal Research: From mice to mammoths

1. CRISPR SYSTEM
PRABESH KOIRALA
PLB-05M-2019
Center for Biotechnology
AFU, Rampur

2. Terminologies
Repeats
Spacer
CAS protein
CRISPR RNA (crRNA)
Trans-activating crRNA (tracrRNA)
Guide RNA (gRNA)
Protospacer adjacent motifs (PAMs)

3. CRISPR System
• CRISPR locus (Clustered Regularly Interspaced Short Palindromic
Repeats)
• Natural immune system of bacteria and archaea,
similar to RNAi mechanism in eukaryotes
Francisco Mojica
Emmanuelle Charpentier Jennifer Doudna
CRISPR Array
CAS operon
Promoter/leader Repeat
Spacer
CAS genes
Zhang et al. (2018). RSC Adv.
Future Nobel Laureates??

4. Actions of CRISPR System
M. Rutkauskas et al. (2017). Methods of Enzymology.
crRNA
Protein complex
2. Expression
1. Adaptation
3. Interference
Pre-crRNA

5. Models of CRISPR System
Makarova et al. (2011). Nat. Rev. Microbio.
cascade

6. Mechanism of DNA Recognition
CAS protein
5’
3’
crRNA-tracrRNA chimera

7. Double Strand Break Repairing
Lui et al. (2019)

8. Tools for Genetic Engineering
DNA structure/Sequencing
Restriction enzymes
PCR
Genome editing(CRISPR-CAS)

9. Applications of CRISPR
Agriculture : USDA approved CRISPR-edited food as safe as ones bred from
conventional way
Gene Therapy & Medicine: Treatment of genetic disease, Cancer, blindness,
Alzheimer’s disease, HIV, etc.
Bio-fuel and Chemicals: increased oil yield from GE strain of ocean algae
Transgenic Animal Research: From mice to mammoths

10. THANK YOU!