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Copy of CRISPR Cas9

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by peng kun on 12 August 2013

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Transcript of Copy 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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