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[DESCRIPTION]
Cas9 Nuclease,&nbsp;<em>S. pyogenes&nbsp;</em>(Cas9), is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA&nbsp;in vitro&nbsp;using Cas9 and a single guide RNA (sgRNA).

[BEFORE_START]
We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found&nbsp;<a href="https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination" target="_blank">here</a>.

[GUIDELINES]
<strong>OVERVIEW</strong>:<br />Cas9 Nuclease,&nbsp;S. pyogenes, (Cas9) is a double-stranded DNA endonuclease that is guided to its target by sequence complementarity of a small RNA loaded into the protein. This protocol describes how to digest double-stranded DNA&nbsp;in vitro&nbsp;using Cas9 and a single guide RNA (sgRNA).&nbsp;<br /><br /><strong>REQUIRED MATERIALS:</strong><br />
<ul style="list-style-type: disc;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Cas9 Nuclease, S. pyogenes (NEB&nbsp;</span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/m0386-cas9-nuclease-s-pyogenes" target="_blank">#M0386</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">)</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">10X Cas9 Nuclease Reaction Buffer</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">Nuclease-free water</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">sgRNA containing the targeting sequence in the region of interest</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">&nbsp;sgRNAs can be generated by in vitro transcription using the HiScribe T7 Quick High-Yield RNA synthesis Kit (NEB&nbsp;</span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/e2050-hiscribe-t7-quick-high-yield-rna-synthesis-kit">#E2050</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">) using linearized plasmid, PCR products, or oligonucleotides as templates</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">sgRNAs must contain sequence complementary to the target DNA (1,2)For information on design of sgRNA transcription templates please visit&nbsp;</span><a class="ga-event" style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="http://www.addgene.org/CRISPR/guide/#Design" target="_blank" data-ga-category="External Link" data-ga-action="https://www.neb.com/protocols/2014/05/01/in-vitro-digestion-of-dna-with-cas9-nuclease-s-pyogenes-m0386" data-ga-label="http://www.addgene.org/CRISPR/guide/#Design">Addgene</a></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">DNA substrate containing the target sequence</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">The substrate DNA can be circular or linearized plasmid, PCR products, or synthesized oligonucleotides</span></li>
</ul>
<br /><strong>OPTIONAL MATERIALS</strong>:<br />Apparatus and reagents for DNA fragment analysis <br />
<ul style="list-style-type: disc;">
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">E. g. Agarose gel electrophoresis apparatus</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">DNA Loading Dye (e.g. Gel Loading Dye, Purple (6X) NEB&nbsp;</span><a style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;" href="https://www.neb.com/products/b7024-gel-loading-dye-purple-6x" target="_blank">#B7024S</a><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">)</span></li>
<li><span style="font-family: 'Open Sans', sans-serif; font-size: 13px; line-height: 20px;">E.g. Agilent Bioanlyzer or similar</span></li>
</ul>
<br /><strong>BEFORE YOU START</strong>:<br />We strongly recommend wearing gloves and using nuclease-free tubes and reagents to avoid RNase contamination. Further recommendations for avoiding ribonuclease contamination can be found&nbsp;<a href="https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination" target="_blank">here</a>.<br />Reactions are typically 30 &mu;l but can be scaled up as needed. Reactions should be assembled in nuclease-free microfuge tubes or PCR strip tubes.<br />It is essential to keep the molar ratio of Cas9 and sgRNA per target site at 10:10:1 or higher to obtain the best cleavage efficiency. A calculator can be found&nbsp;<a href="http://nebiocalculator.neb.com/" target="_blank">here</a>.<br />Prepare 300nM sgRNA by diluting the stock with nuclease-free water on ice.<br />Prepare 30nM substrate DNA with a single target sequence by diluting the stock with nuclease-free water on ice.<br /><br /><strong>PROCEDURE:</strong><br />1. Assemble the reaction at room temperature in the following order:<br />
<table>
<tbody>
<tr class="border">
<td><strong>Component</strong></td>
<td><strong>Volume</strong>&nbsp;(for 30 &micro;l reaction)</td>
</tr>
<tr class="border">
<td>Nuclease-free water</td>
<td>20 &micro;l</td>
</tr>
<tr class="border">
<td>10X Cas9 Nuclease Reaction Buffer</td>
<td>3 &micro;l</td>
</tr>
<tr class="border">
<td>300nM sgRNA</td>
<td>3 &micro;l (30nM final)</td>
</tr>
<tr class="border">
<td>1 &micro;M Cas9 Nuclease, S.pyogenes (<a href="https://www.neb.com/products/m0386-cas9-nuclease-s-pyogenes">M0386S</a>)</td>
<td>1 &micro;l (~30nM final)</td>
</tr>
<tr class="border">
<td>Reaction volume</td>
<td>27 &micro;l</td>
</tr>
<tr class="border"><th colspan="2">Pre-incubate for 10 minutes at 37⁰C</th></tr>
<tr class="border">
<td>30nM substrate DNA</td>
<td>3 &micro;l (3nM final)</td>
</tr>
<tr class="border">
<td>Total reaction volume</td>
<td>30 &micro;l</td>
</tr>
</tbody>
</table>
*The substrate DNA and sgRNA, and nuclease-free water are not included.&nbsp;<br /><br />2. Mix thoroughly and pulse-spin in a microfuge.<br />3. Incubate at 37&deg;C for 1 hour.<br />4. Proceed with fragment analysis.<br /><br /><strong>REFERENCES</strong>:<br />1. Jinek et al. (2012) Science 337 (6096) 816-821.<br />2. Larson et al. (2013) Nature Protocol 8 (2180-2196).<br />3. Mali et al. (2013) Science 339 (6121): 823-826.

[STEPS]
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