CRISPR And Superbugs

Guided by: Dr. Rajnikant G.K.

Made and Presented By : Amay Redkar, B130855BT

Hallmarks of CRISPRs • Adaptive Immunity of

sorts in Bacteria and Archaea

• Provides Defence against bacteriophages

• Shares no phylogenetic relationship with eukaryotic immune system

CRISPR RepeatImage Source: Bolotin et. al. 2005

Fragment from a Viral genome

3 Steps To Acquire Immunity In Bacteria

• Given system was first identified in Streptococcus pyogenes

• Recognition and adaptation

• Expression• Interference

Image Source: Weidenheft et. al. 2012

Cas9 Endonuclease Mechanism

• Protospacer Adjacent Motif (PAM)

• Trans-activating crRNA (tracrRNA)

• CRISPR RNA (crRNA)• Double Strand Breaks(DSBs)

Image Source: Jinek et. al. 2012

Genome Editing and DSBs

Image Source: Khalili et. al. 2015 & Ran et al. 2013

Sgrna - Single Guide RNANHEJ - Non-homologous End-joiningHDR - Homology Directed Repair

Advantages of CRISPR in Genome Editing

• 2 component model for genome engineering cleaving enzyme• Relies on RNA-DNA base pairing for site recognition, instead of

protein.• Cas9 mediated editing is efficient, site-specific and can be multiplexed• Drastically but relatively cheap

Superbug Infections• Widespread can and will be fatal for us as a species• High mortality rate• Resistance can be developed • In cell or• Acquired via Horizontal transfer

• But what is so different?• Mechanism-Pumpers and Chewers • E.g. MRSA, CRE, C. difficile, tuberculosis and Klebsiella• Case -The Nevada Woman

Image Source: Ross et. al. 2014

• RNA Guided Nucleases(RGN)

Advantages of CRISPR as a Antimicrobial Agent

•Unprecedented Specificity, not just narrow spectrum but targeting bugs that have specific genes only

•Development of anti CRISPR genes is not viable for cells because of the extreme competition from phages.