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CRISPR Cas9

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by

Paulo Burke

on 4 March 2013

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Transcript of CRISPR Cas9

CRISPR Systems There are three types of CRISPR/Cas systems (I, II and III). Cas9 Gene: The Main Protein Cas9 is a DNA endonuclease guided by two RNAs (crRNA tracrRNA).
Each Cas9 nuclease domain cleaves one DNA strand.
Cas9 contains domains homologous to both HNH and RuvC endonucleases.
Each Cas9 nuclease domain cleaves one DNA strand. Dual-RNA Requirements Cas9 Motif The PAM conforms to an NGG consensus sequence, containing two G:C base pairs that occur one base pair downstream of the crRNA binding sequence.
Chimeric RNA Cas9 can be programmed using a single engineered RNA molecule combining tracrRNA and crRNA features.
A Programmable Dual-RNA–Guided
DNA Endonuclease in Adaptive
Bacterial Immunity Paulo Burke Martin Jinek,1,2* Krzysztof Chylinski,3,4* Ines Fonfara,4 Michael Hauer,2† Jennifer A. Doudna,1,2,5,6‡ Emmanuelle Charpentier4‡ Types I and III Type II HNH Domain Two pairs of conserved histidines and one asparagine that create a zinc finger domain.
Cleaves dsDNA. Zinc Finger RuvC Domain Cleaves cruciform junctions, ssDNA. Conclusions Cas9 is a dsDNA endonuclease guided by RNA.

Each HNH and RuvC-like domain cleaves one strand of the target DNA.

The system is efficient, versatile, and programmable by changing the DNA target-binding sequence in the guide chimeric RNA.

Alternative methodology based on RNA-programmed Cas9 that could offer considerable potential for gene-targeting and genome-editing applications.
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