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<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=''>
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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'>DOI 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 TracrRNA:crRNA has been replaced by an engineered single guide RNA (sgRNA) that maintains the two main features of the RNA: the complementary 20-nucleotide long sequence at the 5' end and the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.
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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'>DOI 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 ==
== Overall Structure ==
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The cas9 protein complex has a seahorse shaped structure that is composed of 11 cas subunits. Cascade (CRISPR-associated complex for antiviral defense) is from the type-I CRISPR-cas system, and the crystal structure of this surveillance complex gives insight into the overall structure. ('''IMAGE''') The body is comprised of six subunits (Cas7.1-7.6) wrapped around the crRNA in a helical filament with a dimer of Cse2 in the center <ref name='four'>DOI 10.1126/science.1256996</ref>. The head of the Cas7 body is capped by Cas6e and the 3' end of the crRNA while the 5' end and Cas5 cap the tail. The N-terminal end of Cse1 is also at the tail and the C-terminal end contains a bundle of 4 helices that contact Cse2.2. The Cse2 dimer, Cas7 filament, and four-helix bundle of Cse1 form a groove immediately next to guide region of the crRNA. This is where the ssDNA target fits into the complex <ref name='four'/>.
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The Cas9 protein complex has a seahorse shaped structure that is composed of 11 cas subunits. Cascade (CRISPR-associated complex for antiviral defense) is from the type-I CRISPR-cas system, and the crystal structure of this surveillance complex gives insight into the overall structure. The body is comprised of six subunits (Cas7.1-7.6) wrapped around the crRNA in a helical filament with a dimer of Cse2 in the center <ref name='four'>DOI 10.1126/science.1256996</ref>. The head of the Cas7 body is capped by Cas6e and the 3' end of the crRNA while the 5' end and Cas5 cap the tail. The N-terminal end of Cse1 is also at the tail and the C-terminal end contains a bundle of 4 helices that contact Cse2.2. The Cse2 dimer, Cas7 filament, and four-helix bundle of Cse1 form a groove immediately next to guide region of the crRNA. This is where the ssDNA target fits into the complex <ref name='four'/>.
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== Binding Interactions with Target dsDNA ==
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The groove in which the ssDNA target fits is not formed until cas9 undergoes a conformational change upon association with a target dsDNA. The arginine-rich alpha helix to which tracrRNA binds serves as a hinge between the structural lobes of the overall structure. The conformational change is thought to take part in the R-loop formation that unwinds the target dsDNA and allows for interactions between crRNA and its complementary section <ref name='one'/>.
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The groove in which the ssDNA target fits mentioned above is not formed until cas9 undergoes a conformational change upon association with a target dsDNA. The arginine-rich alpha helix to which tracrRNA binds serves as a hinge between the structural lobes of the overall structure. The conformational change is thought to take part in the R-loop formation that unwinds the target dsDNA and allows for interactions between crRNA and its complementary section <ref name='one'/>.
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== DNA Interactions with PAM ==
The PAM sequence has been shown to be critical to inducing DNA binding, as Cas9 is unable to recognize even fully complementary sequences without it <ref name='one'/>. Upon formation of the substrate-protein complex, the nuclease and helical recognition lobes of Cas9 and the target ssDNA form a four-way junction straddling the arginine-rich alpha helix mentioned previously <ref name='five'>DOI 10.1038/nature13579</ref>. Nucleotides on either side of the PAM containing region (-1 to -8 on the target strand and +1 to +8 on the target strand) are base paired, and strand separation occurs only at the first base pair on the target strand (+1). The kink formed from the strand separation places the PAM sequence in a positively-charged groove known as the PAM-interacting domain <ref name='five'/>.
The PAM sequence has been shown to be critical to inducing DNA binding, as Cas9 is unable to recognize even fully complementary sequences without it <ref name='one'/>. Upon formation of the substrate-protein complex, the nuclease and helical recognition lobes of Cas9 and the target ssDNA form a four-way junction straddling the arginine-rich alpha helix mentioned previously <ref name='five'>DOI 10.1038/nature13579</ref>. Nucleotides on either side of the PAM containing region (-1 to -8 on the target strand and +1 to +8 on the target strand) are base paired, and strand separation occurs only at the first base pair on the target strand (+1). The kink formed from the strand separation places the PAM sequence in a positively-charged groove known as the PAM-interacting domain <ref name='five'/>.
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== DNA Interactions with HNH and RuvC Nuclease Domains ==
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<ref name='six'>DOI 10.1016/j.cell.2014.02.001</ref>

Revision as of 02:42, 11 October 2016

Introduction

CRISPR RNA-guided surveillance complex with target ssDNA

Drag the structure with the mouse to rotate

References

  1. ↑ 1.0 1.1 1.2 1.3 1.4 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
  2. ↑ 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
  3. ↑ Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature. 2011 Mar 31;471(7340):602-7. doi: 10.1038/nature09886. PMID:21455174 doi:http://dx.doi.org/10.1038/nature09886
  4. ↑ 4.0 4.1 Mulepati S, Heroux A, Bailey S. Crystal structure of a CRISPR RNA-guided surveillance complex bound to a ssDNA target. Science. 2014 Aug 14. pii: 1256996. PMID:25123481 doi:http://dx.doi.org/10.1126/science.1256996
  5. ↑ 5.0 5.1 Anders C, Niewoehner O, Duerst A, Jinek M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature. 2014 Jul 27. doi: 10.1038/nature13579. PMID:25079318 doi:http://dx.doi.org/10.1038/nature13579
  6. ↑ Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani R, Zhang F, Nureki O. Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell. 2014 Feb 27;156(5):935-49. doi: 10.1016/j.cell.2014.02.001. Epub 2014 Feb, 13. PMID:24529477 doi:http://dx.doi.org/10.1016/j.cell.2014.02.001

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