TITLE: No Title
AUTHORS: 

[DESCRIPTION]
   This protocol describes how to deliver ribonucleoprotein (RNP) complexes that consist of purified Cas9 nuclease duplexed with chemically modified synthetic single guide RNA (sgRNA) to standard immortalized cell lines (adherent or suspension). Although optimized for HEK293 (human embryonic kidney 293 cells), this protocol is applicable to many other cell lines (A549, U2OS, HeLa, CHO, MCF-7). RNP delivery is accomplished using Lipofectamine™ CRISPRMAX™Transfection Reagent. Chemically modified sgRNAs are designed to resist exonucleases and innate intracellular immune cascades that can lead to cell death. Synthego chemically modified synthetic sgRNAs are of exceptional quality and consistently drive high editing efficiencies. Data that demonstrate editing efficiency in HEK293 cells are included.
  
  
 
 
 

[GUIDELINES]
<div title="Page 1">
<div title="Page 4">
<div>
<div>
<p>This protocol is meant to serve as a starting point for lipofection of immortalized cells in a 24-well plate format. It may be necessary to experimentally optimize volumes and ratios for RNP formation for each cell type and for other culture plate formats. It is critical to add reagents in the order recommended in the steps. Prepare the RNP complexes with the Lipofectamine<sup>TM</sup> Cas9 Plus<sup>TM</sup> Reagent and Opti-MEM<sup>TM</sup> I Reduced Serum Medium in a separate tube (Tube 1) before adding diluted CRISPRMAX<sup>TM</sup> Reagent (Tube 2).</p>
<p> </p>
<p>Reaction volumes are for EACH WELL and should be scaled up proportionally for the number of wells to be used.</p>
</div>
</div>
</div>
<p> </p>
<p><em>Abbreviations:</em></p>
<p> </p>
<p>CRISPR: clustered regularly interspaced short palindromic repeats Cas9: CRISPR associated protein 9<br />sgRNA: single guide RNA<br />RNP: ribonucleoprotein</p>
<p>PCR: polymerase chain reaction<br />ICE: inference of CRISPR edits<br />FACS: fluorescence-activated cell sorting TE: Tris EDTA<br />PBS: phosphate-buffered saline<br />GFP: green fluorescent protein</p>
<p> </p>
<div title="Page 2">
<p><strong>Important Considerations</strong></p>
<ul>
<li>
<p>All Synthego and CRISPRMAX<sup>TM</sup> reagents should be stored according to the manufacturer’s recommendations.</p>
</li>
<li>
<p>This protocol was optimized in HEK293 cells and can be used for other common cell lines such as A549, U2OS, HeLa,</p>
<p>CHO, MCF-7.</p>
</li>
<li>
<p>Successful transfection is critically dependent on cell density. It may be necessary to optimize cell seeding densities in</p>
<p>order to determine the most appropriate level of confluence for transfection.</p>
</li>
<li>
<p>For fast growing cells, seed fewer cells. Cell seeding is based on the rate of cell growth. Suggested starting cell numbers</p>
<p>are listed in the protocol below.</p>
</li>
<li>
<p>In order to maximize CRISPR editing, be sure to include trypsinization (<a href="https://www.protocols.io/view/crispr-editing-of-immortalized-cells-with-rnps-usi-srned5e?step=9" target="_blank" rel="noopener noreferrer">Step 9</a>) in order to perform a reverse transfection of RNPs.</p>
</li>
<li>
<p>Use cells at lowest passage number possible.</p>
</li>
<li>
<p>Cas9 nuclease can be diluted in Opti-MEM<sup>TM</sup> I Reduced Serum Medium in order to achieve a working concentration</p>
<p>according to the plate volume.</p>
</li>
<li>
<p>Synthego recommends sgRNA:Cas9 ratio of 1.3:1 for RNP formation. It may be necessary to optimize ratios for different cell lines/conditions.</p>
</li>
<li>
<p>RNP complexes are formed in Opti-MEM<sup>TM</sup> I Reduced Serum Medium and can be added directly to cells in culture medium irrespective of antibiotics. Following transfection, it is not necessary to remove RNP complexes or to add or</p>
<p>change medium.</p>
</li>
</ul>
<p> </p>
<div title="Page 3">
<p><em>Working with RNA and RNPs</em></p>
<ul>
<li>Wearing gloves and using nuclease-free tubes and reagents is recommended in order to avoid RNase contamination. • Always maintain sterile technique and use sterile filter pipette tips.</li>
<li>All Synthego reagents should be stored according to the manufacturer’s recommendations.</li>
<li>Synthetic sgRNA should be dissolved in TE buffer and diluted to a working concentration using nuclease-free water. Please consult the <a href="https://www.synthego.com/wp-content/uploads/2017/05/Quick-Start-Guide-1.3.pdf" target="_blank" rel="noopener noreferrer">Synthego Quick Start Guide</a> for best practices related to dissolving and storing synthetic sgRNAs.</li>
<li>RNP complexes are stable at room temperature for up to 1 hour (may be stored at 4°C for up to one week, or at -20°C for up to 1 month). Note that RNPs stored at 4°C may become susceptible to contamination from microbial growth after long periods of time.</li>
</ul>
<div title="Page 3">
<div>
<div>
<p> </p>
<p><strong>Suggested Controls</strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5twfw26.png" width="803" height="248" data-src="https://s3.amazonaws.com/pr-journal/5tvfw26.png" data-ofn="Screen Shot 2018-08-20 at 10.58.43 AM.png" /></p>
<p> </p>
<div title="Page 3">
<div>
<div>
<p><strong>Timeline</strong></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5tyfw26.png" width="798" height="249" data-src="https://s3.amazonaws.com/pr-journal/5txfw26.png" data-ofn="Screen Shot 2018-08-20 at 10.59.36 AM.png" /></p>
<p> </p>
<div title="Page 3">
<div>
<div>
<p><em><strong>Note:</strong> cell seeding may take 1-2 days and incubation after transfection may take 2-3 days.</em></p>
<p> </p>
<p> </p>
<div title="Page 6">
<div>
<div>
<p><strong>Additional Information</strong></p>
<p> </p>
<p>For an up-to-date list of all Synthego Protocols and other resources, please visit <a href="https://www.synthego.com/resources" target="_blank" rel="noopener noreferrer">synthego.com/resources</a></p>
<p> </p>
<p>For technical assistance, contact our Scientific Support Team:</p>
<p>Ph: 888.611.6883</p>
<p>Email: <a href="mailto:support@synthego.com" target="_blank" rel="noopener noreferrer">support@synthego.com</a></p>
</div>
</div>
</div>
<p> </p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
SECTION: Pre-Lipofection -  Seed cells
?. 
SECTION: Setup & Lipofection - Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
?. 
SECTION: Setup & Lipofection - Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
?. 
SECTION: Setup & Lipofection - Assemble RNP Complexes (1.3:1 sgRNA to Cas9 ratio)
?. 
SECTION: Setup & Lipofection - Prepare Transfection Solution
?. 
SECTION: Setup & Lipofection - Prepare Transfection Solution
?. 
SECTION: Setup & Lipofection - Make RNP-Transfection Solution
?. 
SECTION: Setup & Lipofection - Make RNP-Transfection Solution
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Prepare Cells
?. 
SECTION: Setup & Lipofection - Transfect Cells
?. 
SECTION: Setup & Lipofection - Transfect Cells
?. 
SECTION: Post-Lipofection - Analysis
?. 
SECTION: Post-Lipofection - Analysis
?.