TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cas9 nuclease may be used <em>in vivo</em> to create targeted genome modifications.  There are several ways in which to introduce Cas9-guide RNA complexes into cells.  Here we present a method for the transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX.  This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.</p>

[BEFORE_START]
<ul>
<li>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 here: <a href="https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination" target="_blank">https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination</a></li>
<li>Transfection conditions may be highly variable.  It is recommended to optimize your conditions for each cell type and Cas9 target you may have.  This protocol follows conditions that have been optimized for a particular target and use of HEK293 cells.</li>
</ul>

[GUIDELINES]
<p><strong>Overview:</strong></p>
<p> </p>
<p>Cas9 nuclease may be used <em>in vivo</em> to create targeted genome modifications.  There are several ways in which to introduce Cas9-guide RNA complexes into cells.  Here we present a method for the transfection of Cas9 RNP’s into HEK293 FT cells using Thermo Fisher Lipofectamine® RNAiMAX.  This is a ‘reverse transfection’ method that uses a final concentration of 10 nM RNP per transfection in a 96-well culture plate.</p>
<p> </p>
<p><strong>Required Materials:</strong></p>
<p> </p>
<p><span style="text-decoration: underline;">Cell Culture and Transfection</span></p>
<ul>
<li>HEK293 cells (or other cell line) at 70-90% confluency in a T-75 flask.</li>
<li>EnGen™ Cas9 Nuclease NLS, <em>S. pyogenes</em> (<a href="https://www.neb.com/products/m0646-engen-cas9-nls-s-pyogenes" target="_blank">M0646T or M0646M</a>)</li>
<li>sgRNA containing the targeting sequence in the region of interest</li>
<ul>
<li>sgRNAs can be generated using the EnGen™ sgRNA Synthesis Kit, <em>S. pyogenes</em> (<a href="https://www.neb.com/products/e3322-engen-sgrna-synthesis-kit-s-pyogenes" target="_blank">E3322S</a>).</li>
<li>sgRNAs must contain the target sequences (20 nucleotides) adjacent to the Protospacer Adjacent Motif (PAM, NGG) in the target DNA. (1,2).  See the EnGen sgRNA Synthesis Kit <a href="https://www.neb.com/~/media/Catalog/All-Products/E1EB7E7B31C64D9DBC8D7DAFEEF0C8D7/Datacards%20or%20Manuals/manualE3322.pdf" target="_blank" data-ga-category="Download: PDF" data-ga-action="Download" data-ga-label="https://www.neb.com/~/media/Catalog/All-Products/E1EB7E7B31C64D9DBC8D7DAFEEF0C8D7/Datacards%20or%20Manuals/manualE3322.pdf">manual</a> for further details.</li>
</ul>
<li> Lipofectamine RNAiMAX Transfection Reagent (ThermoFisher)</li>
<li>Sterile 1X PBS without Ca2+ and Mg2+</li>
<li>DMEM with Glutamax (or appropriate growth medium) with 10% FBS</li>
<li>Optimem Reduced Serum Medium (ThermoFisher)</li>
<li>96-well culture plate</li>
</ul>
<p><span style="text-decoration: underline;">DNA Extraction and Genome Editing Analysis</span> </p>
<ul>
<li>EnGen™ Mutation Detection Kit (<a href="https://www.neb.com/products/e3321-engen-mutation-detection-kit#tabselect0" target="_blank">E3321S</a>)</li>
<li>Epicentre QuickExtract™ DNA Extraction Solution (Epicentre #QE09050)</li>
</ul>
<p><strong>Before You Start:</strong></p>
<ul>
<li>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 here: <a href="https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination" target="_blank">https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination</a>.</li>
</ul>
<ul>
<li>Transfection conditions may be highly variable. It is recommended to optimize your conditions for each cell type and Cas9 target you may have. This protocol follows conditions that have been optimized for a particular target and use of HEK293 cells.</li>
</ul>

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