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Cas9 cleaves each of the target DNA strands a few nucleotides upstream of the PAM (red arrowheads). Each strand is cleaved by a different nuclease domain present in Cas9 (HNH and RuvC domains). These domains have been mutated independently to generate Cas9 nickases. | Cas9 cleaves each of the target DNA strands a few nucleotides upstream of the PAM (red arrowheads). Each strand is cleaved by a different nuclease domain present in Cas9 (HNH and RuvC domains). These domains have been mutated independently to generate Cas9 nickases. | ||
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Latest revision as of 07:30, 14 February 2020
CRISPR Cas9 interaction with target DNA
The CRISPR/Cas9 complex of Streptococcus pyogenes consists of the Cas9 protein (in gray) and a guide RNA that is a chimera of natural crRNA and tracRNA (in orange).
The targeting sequence at the 5′end of the guide RNA base-pairs with complementary sequences on the target DNA (in blue); the targeting sequence is 20 nucleotides long, but may be shortened to increase specificity (the addition of 1 to 2 unpaired nucleotides at the 5′ end is also tolerated).
The presence of a PAM (protospacer adjacent motif, NGG for Streptococcus pyogenes), located immediately downstream of the 20-nucleotide sequence targeted by the guide RNA, is also essential for target recognition and cleavage.
The PAM sequence does not have a counterpart on the guide RNA. Following recognition of the PAM and base-pairing between the guide RNA and the target,
Cas9 cleaves each of the target DNA strands a few nucleotides upstream of the PAM (red arrowheads). Each strand is cleaved by a different nuclease domain present in Cas9 (HNH and RuvC domains). These domains have been mutated independently to generate Cas9 nickases.
Reference
Gilles AF & Averof M. (2014). Functional genetics for all: engineered nucleases, CRISPR and the gene editing revolution. Evodevo , 5, 43. PMID: 25699168 DOI.
Copright
© 2014 Gilles and Averof; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Gilles and Averof EvoDevo 2014 5:43 doi:10.1186/2041-9139-5-43 Text above from original figure legend.
2041-9139-5-43-2.jpg
Cite this page: Hill, M.A. (2024, April 19) Embryology CRISPR Cas9 interaction with target DNA.jpg. Retrieved from https://embryology.med.unsw.edu.au/embryology/index.php/File:CRISPR_Cas9_interaction_with_target_DNA.jpg
- © Dr Mark Hill 2024, UNSW Embryology ISBN: 978 0 7334 2609 4 - UNSW CRICOS Provider Code No. 00098G
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