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

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

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

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.

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

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.

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

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

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

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

Future applications Source: Doudna y Charpentier, 2014

Bibliography Bhaya, D., Davison, M., Barrangou, R. 2011. CRISPR-Cas System in Bacteria and Archaea: Versatile Small RNAs for Adaptative Defense and Regulation. Annual Review of Genetics. 45:273-297. Bortesi, L., and Fischer, R. 2014. The CRISPR/Cas9 system for plant genome editing and beyond. Biotechnol. Adv. 33: 41–52. Doudna, J.A., and Charpentier, E. 2014. The new frontier of genome engineering with CRISPR-Cas9. Science. 346: 1258096–1258096. Horvath,P., Barrangou,R. 2010. CRISPR/Cas, the immune System of Bacteria and Archaea. Science: 327: 167-170. Pennisi, E. 2013. The CRISPR Craze. Science. 341: 833-836.