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 introduction of Cas9 RNP’s into HEK293 FT cells using the Thermo Fisher Neon Electroporation System.  This method uses a final concentration of 20 nM RNP per transfection in a 24-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>Please refer to the Neon Transfection System manual for proper usage of the equipment.</li>
</ul>

[GUIDELINES]
<p><strong>Overview:</strong></p>
<p> </p>
<p>Cas9 nuclease may be used in vivo 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 introduction of Cas9 RNP’s into HEK293 FT cells using the Thermo Fisher Neon Electroporation System. This method uses a final concentration of 20 nM RNP per transfection in a 24-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>ThermoFisher Neon Transfection System 10 µl Kit (MPK1025)</li>
<li>Sterile 1X PBS without Ca2+ and Mg2+</li>
<li>DMEM with Glutamax (or appropriate growth medium) with 10% FBS</li>
<li>24-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>
<p> </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>Please refer to the Neon Transfection System manual for proper usage of the equipment.</li>
</ul>

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