User:Wally Novak/Sandbox Brown
From Proteopedia
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== Introduction == | == Introduction == | ||
<StructureSection load='4qyz' size='340' side='right' caption='CRISPR RNA-guided surveillance complex with target ssDNA' scene=''> | <StructureSection load='4qyz' size='340' side='right' caption='CRISPR RNA-guided surveillance complex with target ssDNA' scene=''> | ||
| - | Cas9 is a large multifunctional protein that plays a central role in the CRISPR-Cas adaptive defense mechanism found in a vast amount of bacteria and archaea <ref name='one'>DOI 10.1126/science.1258096</ref>. It accomplishes this through the use of antisense RNAs which serve as signatures from past viral invasions <ref>DOI 10.1186/1745-6150-1-7</ref>. The adaptive immunity occurs in three stages: insertion of invading DNA into CRISPR locus, transcription of precursor crRNA from CRISPR locus that will be used to generate crRNA that matches its target sequence for 20 | + | Cas9 is a large multifunctional protein that plays a central role in the CRISPR-Cas adaptive defense mechanism found in a vast amount of bacteria and archaea <ref name='one'>DOI 10.1126/science.1258096</ref>. It accomplishes this through the use of antisense RNAs which serve as signatures from past viral invasions <ref>DOI 10.1186/1745-6150-1-7</ref>. The adaptive immunity occurs in three stages: insertion of invading DNA into CRISPR locus, transcription of precursor crRNA from CRISPR locus that will be used to generate crRNA that matches its target sequence for 20 nucleotides, and crRNA-directed cleavage of foreign nucleic acids by cas9. PAM (protospacer adjacent motif) sequences must be present adjacent to the crRNA-targeted sequence to be cleaved <ref name='one'/>. In addition to the crRNA, cas9 incoporates another RNA chain that serves to anchor the crRNA to the protein. This tracrRNA is partially complimentary to a piece of the crRNA and interacts with an arginine-rich alpha helix to anchor both pieces of RNA to cas 9 <ref name='three'>DOIdoi:10.1038/nature09886</ref>. Just in the last few years, this defensive mechanism and the cas9 protein has been used to develop genome engineering applications. TracrRNA:crRNA has been replaced by an engineered single guide RNA (sgRNA) that maintains the two main features of the RNA: the complementary 20-nucleotide long sequence at the 5' end and the double-stranded anchor at the 3' end to bind to cas9 <ref name='one'/>. The programmable cas9 protein is then used to create double-stranded breaks in genomic DNA, at which points the genetic sequence could then be altered. |
| - | + | == Overall Structure == | |
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| - | [[Image:cas9image.jpg]] | ||
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| - | == Overall Structure == | ||
The Cas9 protein contains two nuclease domains, HNH and RuvC | The Cas9 protein contains two nuclease domains, HNH and RuvC | ||
Revision as of 00:32, 11 October 2016
Introduction
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References
- ↑ 1.0 1.1 1.2 Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014 Nov 28;346(6213):1258096. doi: 10.1126/science.1258096. PMID:25430774 doi:http://dx.doi.org/10.1126/science.1258096
- ↑ Makarova KS, Grishin NV, Shabalina SA, Wolf YI, Koonin EV. A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted enzymatic machinery, functional analogies with eukaryotic RNAi, and hypothetical mechanisms of action. Biol Direct. 2006 Mar 16;1:7. PMID:16545108 doi:http://dx.doi.org/10.1186/1745-6150-1-7
- ↑ DOIdoi:10.1038/nature09886
