1. Seminar on: CRISPR/Cas9
Institute of Molecular Biology and Biotechnology
INTERUNIVERSITY PROGRAMME MOLECULAR BIOLOGY (IPMB)
Seminar on: CRISPR/Cas9
By: Nateneal Tamerat
and
Wegen Beyene
December 2016
Brussels, Belgium

2. Outline Introduction Historical Background Principle
Current and Prospective Application
Socio-ethical and Ecological Concerns

3. Introduction
CRISPR- Cas9 : is clustered, regularly interspaced, short palindromic repeat and the associated Cas9 protein is naturally an adaptive immunity mechanism in prokaryotes.
CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea.
Cas 9 is an endonuclease, first identified from Streptococcus pyogenes bacteria. It’s genes are often located next to CRISPR repeat-spacer arrays.
Specificity of CRISPR-Cas9 depends on the presence of a sequence- specific Protospacer Adjacent Motif (PAM) and target sequence (20 bases).
Absence of PAM in host genome enable to avoid self-cleavage.
Remember the job of Cas9 is to generate DSB in target sequence and incorporate and store it as memory to combat future bacteriophage invasion.
And that’s why it called an Adaptive immunity system.

4. Introduction
Unlike previous gene editing tools like TALEN, Zink finger and Meganuclease which needs heavy protein engineering, CRISPR- Cas9 is based on programmable RNA guide system. Its easier to apply and inexpensive.
Mega nucleases, ZFN (Zinc Finger Nuclease), and TALEN (Transcription Activator-Like Effector Nuclease) are earlier gene editing tool via DSB generation.
They are inefficiency, time-consuming and labor intensive features compared to CRISPR/Cas9.
TALEN and ZFN are restriction enzyme.

5. Components of CRISPER-Cas9 System
Introduction
Components of CRISPER-Cas9 System
Cas 9
CRISPR-RNA (crRNA) and trans-activating crRNA (tracrRNA).
Protospacer adjacent motif (PAM) is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
*CRISPR/C2c2 from the bacterium Leptotrichia shahii is RNA-guided CRISPR system that targets RNA rather than DNA, and can either cleave single-stranded RNA targets or knock them down.
Host Genome
Bacteriophage Genome

6. Historical Background
Jennifer Doudna
Emmanuelle Charpentier
In 2005 three different work by Bolotin et al, Mojica et al and Pourcel et al reported derivation of CRISPR spacers from bacteriophages and conjugative plasmids genetic elements and its association with recombination genes, in addition, host doesn't get infected by these foreign DNA implying a relationship between CRISPR and immunity against targeted DNA.
Naturally the system consist separate tracr RNA and crRNA, however, Doudna and Charpentier simplified it by combining the two RNA into a single-guide RNA chimera via linker loop. Most importantly, they discover the function of Cas9 and proved that CRISPER-Cas9 is programmable system.

7. The Principle Behind

8. The Principle Behind…

9. Three Stages of CRISPR-Cas9
The Principle Behind…
Three Stages of CRISPR-Cas9
Stage I: Acquisition- DNA fragments of invading phages are incorporated into the host CRISPR locus as spacers between crRNA repeats.
Stage II: Expression/Biogenesis- Cas proteins are expressed, the CRISPR array containing acquired spacers is transcribed into pre-crRNA, and then cleaved into mature crRNAs.
Stage III: Cleavage/Interference- Cas proteins recognize the appropriate target with the guidance of the crRNA and mediate the cleavage of the invading genome to generate DSB at 3 bp upstream of PAM sequence.

10. The Principle Behind…

11. The Principle Behind… Double Strand Break is generated, then What???
Check jurgen notes?
Donor DNA

12. The Principle Behind…
Creating transgenic mice involves harvesting fertilized eggs and electroporating them to insert the CRISPR-Cas9 molecule, then implanting the edited embryos in another mouse. The targeted gene is linked with a gene that produces white fur, making it easy to see which mice have been successfully edited.
*Ribonucleoprotein,
Human Embryo manipulation is …….
CCR5
Fan’s team used CRISPR–Cas9 genome editing to introduce into some of the embryos a mutation that cripples an immune-cell gene called CCR5. Some humans naturally carry this mutation (known as CCR5Δ32) and they are resistant to HIV, because the mutation alters the CCR5 protein in a way that prevents the virus from entering the T cells it tries to infect.
The wide

13. CRISPR/Cas9 Current and Prospective Application
Gene Therapy and Other Therapeutic
Gene Function Study
Agriculture
GMO food
Current and Prospective Application
Disease Vector Control
Biofuel Industry
Enhancement Gene Edition
Organ Transplantation
Animal Disease Model

14. Current and Prospective Application…
Gene Function Study
Gene Therapy and Other Therapeutic
Organ Transplant
Animal Disease Model

15. Current and Prospective Application…
Enhancement Genome editing
GMO food
Microbial Biofuel
Disease Vector Gene Modification

16. Socio-ethical and Ecological Concerns
CRISPR-cas9 revives many previous gene manipulation related social, ethical and ecological concerns with its relatively simple construction and low cost.
International regulatory means and clinical trial guidelines for the possible use of this technique in germline genome edition and genetic enhancement.
Through risk assessment on its environmental impact should be conducted to avoid ecological impairment.
Further research and public dialogue over the social, ethical and environmental implications with the need of regulation should be serious considered.

17. THANK YOU

18. References
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA and Charpentier E., , A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337: Pourcel C, Salvignol G, and Vergnaud G., CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151(Pt 3): Mojica FJ1, Díez-Villaseñor C, García-Martínez J, Soria E., Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. J Mol Evol, 60(2): Bolotin A1, Quinquis B, Sorokin A, Ehrlich SD., Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology 151(Pt 8): Carmen, B Emerging gene editing strategies for Duchenne muscular dystrophy targeting stem cells. Review: Frontiers in Physiology. 148 (5):1- 7. Ding Y , Li H, Chen L.L and Xie K, Recent Advances in Genome Editing Using CRISPR/Cas9. Front. Plant Sci,7:1-12. Martina Baumann., CRISPR/Cas9 genome editing – new and old ethical issues arising from a revolutionary technology. Nanoethics,10:139–159. Faster, more efficient CRISPR editing in mice by Robert Sander. Accessed on 15/12/2016. Principles and Applications of CRISPR/Cas9 System. Accessed on 15/12/2016. Jennifer Doudna (UCBerkeley/HHMI): Genome Engineering with CRISPR-Cas9. Accessed on 14/12/2016.