1. CRISPR/Cas9 GENOMICS Lucía Álvarez González
Master in Advanced Genetics
Universitat Autònoma de Barcelona

2. Introduction 2000 CRISPR Francisco J. M. Mojica
Source: Doudna y Charpentier, 2014

3. Introduction Objective
To explain the methodology of the CRISPR system, both as an adaptive inmune system in microorganisms and as a genome editing technique.
Objective
CRISPR as an adaptive immune system in archaea and bacteria.
CRISPR as a specific and directed genome editing technique.
Future of the technique

4. CRISPR in microorganism
CRISPR as an adaptive immune system in archaea and bacteria.
CRISPR in microorganism
Immune adaptive system
Clustered Regularly Interspaced Short Palindromic Repeats
The functioning of this mechanism is based on the ability of bacteria to recognize the genetic material of viruses and plasmids that have previously infected them (both they and their predecessors) and generate cuts in their genetic material thus preventing infection.

5. CRISPR as an adaptive immune system in archaea and bacteria.
Parts of CRISPR system
Cas 9 protein
CRISPR locus
PAM sequence
Source: Horvath y Barrangou, 2010
Source: Doudna y Charpentier, 2014

6. CRISPR as an adaptive immune system in archaea and bacteria.
How the system works
Source: Bhaya et al., 2012
ACQUISITION
The insertion of a short sequence of the invading DNA as a spacer in the CRISPR cluster
EXPRESSION
New attack, the cluster is transcribed with the integrated spacer (pre-crRNA) that matures when it binds to the tracrRNA of the Cas9 protein.
INTERFERENCE
The crRNA recognizes the invader thanks to the PAM sequence and the Cas9 protein, to which it is bound by the tracrRNA, cuts the invading DNA.

7. CRISPR as a specific and directed genome editing technique
Parts of CRISPR/Cas9 system
Cas9
sgRNA
5’ end sgRNA  crRNA
3’ end sgRNA  tracrRNA
Source: Xie y Yang, 2013
Source: Bortesi y Fischer, 2014

8. CRISPR as a specific and directed genome editing technique
Modified Cas9: Cas9 Nickase
Cas9 Nuclease
Cas9 Nickase
Problem: Off-target
Source: Schiml y Puchta, 2016

9. CRISPR as a specific and directed genome editing technique
Modified Cas9: dCas9
Source: Bortesi y Fischer, 2014

10. Specific Vectors Multiplexing Cas9 sgRNA Several sgRNAs in a vector
CRISPR as a specific and directed genome editing technique
Specific Vectors
Multiplexing
Several sgRNAs in a vector
Multiple modifications in the genome in a single generation
Cas9
RNA polimerase II (Pol II) promoter.
sgRNA
RNA polimerase III (Pol III) promoter.
Source: Xie y Yang, 2013

11. Future applications
Source: Doudna y Charpentier, 2014

12. Bibliography
Bhaya, D., Davison, M., Barrangou, R CRISPR-Cas System in Bacteria and Archaea: Versatile Small RNAs for Adaptative Defense and Regulation. Annual Review of Genetics. 45:
Bortesi, L., and Fischer, R The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33: 41–52.
Doudna, J.A., and Charpentier, E The new frontier of genome engineering with CRISPR-Cas9. Science. 346: –
Horvath,P., Barrangou,R CRISPR/Cas, the immune System of Bacteria and Archaea. Science: 327:
Pennisi, E The CRISPR Craze. Science. 341: