User:Wally Novak/Sandbox Brown

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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 begins 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 begins 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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The 5'-NGG- 3' PAM with the trinucleotide on the non-target strand is crucial for loading target DNA into Cas9, as the guanine bases participate in base-specific hydrogen bonding with <scene name='74/744130/Pam/2'>Arg 1333 and Arg 1335</scene> <ref name='five'/>. The residues are extended into the major groove by a beta-hairpin in the C-terminal domain of Cas9. These two arginines have been mutated to alanine residues to test the necessity of the formed hydrogen bonds. Upon substitution of either one, the target DNA binding ''in vitro'' was substantially reduced <ref name='five'/>. The same NGG trinucleotide is not required in the complementary target strand because the target-strand nucleotides complementary to the PAM sequence are not recognized by major groove interactions. This explains why some mismatches are tolerated in the PAM sequence, as long as the guanine residues are present in the non-target strand. The Cas9 sequence motif that contains the needed arginine residues has been found in various species with type-II A Cas9 <ref name='five'/>.
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The 5'-NGG- 3' PAM with the trinucleotide on the non-target strand is crucial for loading target DNA into Cas9, as the guanine bases participate in base-specific hydrogen bonding with <scene name='74/744130/Pam/2'>Arg 1333 and Arg 1335</scene> <ref name='five'/>. The residues are extended into the major groove by a beta-hairpin in the C-terminal domain of Cas9. These two arginines have been mutated to alanine residues to test the necessity of the formed hydrogen bonds. Upon substitution of either one, the target DNA binding ''in vitro'' is substantially reduced <ref name='five'/>. The same NGG trinucleotide is not required in the complementary target strand because the target-strand nucleotides complementary to the PAM sequence are not recognized by major groove interactions. This explains why some mismatches are tolerated in the PAM sequence, as long as the guanine residues are present in the non-target strand. The Cas9 sequence motif that contains the needed arginine residues has been found in various species with type-II A Cas9 <ref name='five'/>.
The minor groove of the target DNA and PAM sequence interacts with Ser 1136 through a water-mediated hydrogen bond <ref name='five'/>. The interaction helps orient the target DNA so that it can base pair with sgRNA/crRNA. The +1 phosphate in the target DNA strand forms hydrogen bonds with Glu 1108 and Ser 1109 through its oxygen atoms. This helps form the phosphate lock loop, allowing the guide RNA to begin base pairing with the target RNA <ref name='five'/>.
The minor groove of the target DNA and PAM sequence interacts with Ser 1136 through a water-mediated hydrogen bond <ref name='five'/>. The interaction helps orient the target DNA so that it can base pair with sgRNA/crRNA. The +1 phosphate in the target DNA strand forms hydrogen bonds with Glu 1108 and Ser 1109 through its oxygen atoms. This helps form the phosphate lock loop, allowing the guide RNA to begin base pairing with the target RNA <ref name='five'/>.
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== DNA Interactions with HNH and RuvC Nuclease Domains ==
== DNA Interactions with HNH and RuvC Nuclease Domains ==
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The HNH nuclease domain is responsible for cleaving the DNA strand complementary to the RNA guide. The <scene name='74/744130/Hnh/1'>active site</scene> is composed of a beta-beta-alpha metal fold made up of three secondary structures and a magnesium ion <ref name='six'>DOI 10.1016/j.cell.2014.02.001</ref>. The important residues include Asn 863, Asp 839, His 840, and Asn 854.The 3'-5' phosphate bond is cleaved by a water molecule activated by the histidine residue. The oxygen from the water performs a nucleophilic attack on the phosphate,as the magnesium ion is coordinated with the phosphate, making it more electrophilic. The other three active site residues coordinate with the magnesium ion as well, using their side chains <ref name='six'/>.
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The HNH nuclease domain is responsible for cleaving the DNA strand complementary to the RNA guide. The <scene name='74/744130/Hnh/1'>active site</scene> is composed of a beta-beta-alpha metal fold made up of three secondary structures (forming a super secondary structure) and a magnesium ion <ref name='six'>DOI 10.1016/j.cell.2014.02.001</ref>. The important residues include Asn 863, Asp 839, His 840, and Asn 854.The 3'-5' phosphate bond is cleaved by a water molecule activated by the histidine residue. The oxygen from the water performs a nucleophilic attack on the phosphate,as the magnesium ion is coordinated with the phosphate, making it more electrophilic. The other three active site residues coordinate with the magnesium ion as well, using their side chains <ref name='six'/>.
The RuvC nuclease domain is responsible for cleaving the DNA strand not complementary to the RNA guide. This nuclease contains a <scene name='74/744130/Ruvc/1'>RNase H fold structure</scene>, which is unsurprising since it is responsible for cleaving single-stranded DNA <ref name='six'/>. The active site residues include His 983, Asp 10, Asp 986, and Glu 762. Mutation of any of these residues results in loss of catalytic function <ref name='six'/>. The mechanism of DNA cleavage is similar to that of the HNH domain, with the His residue activating a water molecule for nucelophilic attack and the side chains of the other three residues coordinating with the magnesium ion <ref name='six'/>.
The RuvC nuclease domain is responsible for cleaving the DNA strand not complementary to the RNA guide. This nuclease contains a <scene name='74/744130/Ruvc/1'>RNase H fold structure</scene>, which is unsurprising since it is responsible for cleaving single-stranded DNA <ref name='six'/>. The active site residues include His 983, Asp 10, Asp 986, and Glu 762. Mutation of any of these residues results in loss of catalytic function <ref name='six'/>. The mechanism of DNA cleavage is similar to that of the HNH domain, with the His residue activating a water molecule for nucelophilic attack and the side chains of the other three residues coordinating with the magnesium ion <ref name='six'/>.

Current revision

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 5.2 5.3 5.4 5.5 5.6 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. ↑ 6.0 6.1 6.2 6.3 6.4 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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Wally Novak

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