Name: Auteur in de schijnwerpers: Efficiënte CRISPR/Cas9-genoombewerking in van beenmerg afgeleide macrofagen voor nauwkeurige genverstoring
Uploaded: 2023-08-04T14:50:00
Description: Watch this Scientific Journal Video about Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption at JoVE.com

Dit werk werd gefinancierd door de NIH-subsidie 5R01AI144149. De schematische figuren zijn gemaakt met BioRender.

1. sgRNA-ontwerp
OPMERKING: Deze stap beschrijft de selectie van de doelsequenties en het ontwerp van de sgRNA's. Het is nuttig om handleidingen te ontwerpen die zich in het eerste grote coderende exon bevinden, zodat elk vertaald eiwit vroeg in het open leesframe wordt verstoord. Het is ook nuttig om doelsequenties te selecteren die binnen hetzelfde exon liggen, omdat dit de analyse van de bewerkingsefficiëntie stroomlijnt (stap 6). De voorbeelden van genoombewerking die bij d...

Een gids voor nauwkeurige genverstoring in primaire beenmerg-afgeleide macrofagen

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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+suppression+of+gene+expression+by+RNAi+in+mammalian+cells.">Stable suppression of gene expression by RNAi in mammalian cells.</a> Proceedings of the National Academy of Sciences. 99 (3), 1443-1448 (2002).</li><li>Laugel, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+isogenic+cells+deficient+for+MR1+with+a+CRISPR/Cas9+lentiviral+system:+Tools+to+study+microbial+antigen+processing+and+presentation+to+human+MR1-restricted+T+cells.">Engineering of isogenic cells deficient for MR1 with a CRISPR/Cas9 lentiviral system: Tools to study microbial antigen processing and presentation to human MR1-restricted T cells.</a> The Journal of Immunology. 197 (3), 971-982 (2016).</li><li>Andreu, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+macrophages+and+J774+cells+respond+differently+to+infection+with+Mycobacterium+tuberculosis.">Primary macrophages and J774 cells respond differently to infection with Mycobacterium tuberculosis.</a> Scientific Reports. 7, 42225 (2017).</li><li>Roberts, A. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cas9++conditionally-immortalized+macrophages+as+a+tool+for+bacterial+pathogenesis+and+beyond.">Cas9+ conditionally-immortalized macrophages as a tool for bacterial pathogenesis and beyond.</a> eLife. 8, e45957 (2019).</li><li>Oberdoerffer, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+of+RNA+interference+in+the+mouse+hematopoietic+system+varies+between+cell+types+and+developmental+stages.">Efficiency of RNA interference in the mouse hematopoietic system varies between cell types and developmental stages.</a> Molecular and Cellular Biology. 25 (10), 3896-3905 (2005).</li><li>Ma, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=YTHDF2+orchestrates+tumor-associated+macrophage+reprogramming+and+controls+antitumor+immunity+through+CD8++T+cells.">YTHDF2 orchestrates tumor-associated macrophage reprogramming and controls antitumor immunity through CD8+ T cells.</a> Nature Immunology. 24 (2), 255-266 (2023).</li><li>Cong, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+genome+engineering+using+CRISPR/Cas+systems.">Multiplex genome engineering using CRISPR/Cas systems.</a> Science. 339 (6121), 819-823 (2013).</li><li>Mali, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339 (6121), 823-826 (2013).</li><li>Cho, S. W., Kim, S., Kim, J. M., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+genome+engineering+in+human+cells+with+the+Cas9+RNA-guided+endonuclease.">Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease.</a> Nature Biotechnology. 31 (3), 230-232 (2013).</li><li>Jinek, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-programmed+genome+editing+in+human+cells.">RNA-programmed genome editing in human cells.</a> eLife. 2, e00471 (2013).</li><li>Kim, S., Kim, D., Cho, S. W., Kim, J., Kim, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+RNA-guided+genome+editing+in+human+cells+via+delivery+of+purified+Cas9+ribonucleoproteins.">Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.</a> Genome Research. 24 (6), 1012-1019 (2014).</li><li>Lin, S., Staahl, B. T., Alla, R. K., Doudna, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+homology-directed+human+genome+engineering+by+controlled+timing+of+CRISPR/Cas9+delivery.">Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery.</a> eLife. 3, 04766 (2014).</li><li>Brinkman, E. K., Chen, T., Amendola, M., van Steensel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Easy+quantitative+assessment+of+genome+editing+by+sequence+trace+decomposition.">Easy quantitative assessment of genome editing by sequence trace decomposition.</a> Nucleic Acids Research. 42 (22), e168 (2014).</li><li>Craft, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR-Cas9-mediated+genome+editing+in+primary+murine+bone+marrow-derived+macrophages.">CRISPR-Cas9-mediated genome editing in primary murine bone marrow-derived macrophages.</a> University of California, Davis. , (2022).</li><li>Herb, M., Farid, A., Gluschko, A., Kr&#246;nke, M., Schramm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+transfection+of+primary+macrophages+with+in+vitro+transcribed+mRNA.">Highly efficient transfection of primary macrophages with in vitro transcribed mRNA.</a> Journal of Visualized Experiments. (153), e60143 (2019).</li><li>Yu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+delivery+of+Cas9+protein/gRNA+complexes+using+lipofectamine+CRISPRMAX.">Improved delivery of Cas9 protein/gRNA complexes using lipofectamine CRISPRMAX.</a> Biotechnology Letters. 38 (6), 919-929 (2016).</li></ol>

Genoombewerking met behulp van geëlektropoleerde Cas9-sgRNA-complexen maakt een effectieve verstoring van de genfunctie in BMDM's mogelijk. De bewerkingsefficiëntie varieert afhankelijk van de doelsequentie en het gen. Doorgaans worden over het algemeen vier tot vijf sgRNA's gescreend om er een te identificeren die zeer actief is. Sommige loci hebben een lagere bewerkingsefficiëntie, hoogstwaarschijnlijk vanwege de chromatinestructuur. In deze gevallen kunnen verschillende wijzigingen worden aangebracht om de bewerkin...

Macrofagen zijn aangeboren immuuncellen die een cruciale rol spelen bij weefselherstel en immuniteit 1,2. Onsterfelijke macrofaagcellijnen, zoals RAW 264.7-cellen van muizen of menselijke THP-1-cellen, hebben verschillende gunstige eigenschappen, waaronder robuuste groei en gemakkelijke genverstoring door vectoren te leveren voor RNA-interferentie of CRISPR/Cas9 3,4. Oncogene transformatie verandert echter hun fysiologie drastisch, wat resulteert in de afwijkende activering van sommige paden en gedempte reacties van andere 5,6. Primaire beenmerg-afgeleide macrofagen (BMDM's) recapituleren nauwer in vivo macrofaagfysiologie, maar blijven een uitdaging om genetisch te manipuleren vanwege de lage efficiëntie van zowel plasmidetransfectie als virale transductie in deze primaire immuuncellen 7,8. Er zijn dus efficiëntere methoden nodig om de genfunctie te verstoren.
CRISPR/Cas9-genoombewerking is een krachtig hulpmiddel voor genetische manipulatie in een reeks biologische systemen, waaronder zoogdiercellen 9,10,11,12. Het Streptococcus pyogenes Cas9-eiwit splitst efficiënt en specifiek dubbelstrengs DNA wanneer het wordt gecomplexeerd met een sequentiespecifiek gids-RNA. DNA-reparatie door de niet-homologe eindverbinding (NHEJ) van het gespleten DNA resulteert in kleine inserties of deleties (indels) die frameshift-mutaties veroorzaken. In vroege studies werden Cas9 en sgRNA's afgeleverd via plasmide- of lentivirale vectoren, die effectieve toedieningsmethoden zijn voor veel cellijnen 9,10. Primaire cellen en in het bijzonder primaire immuuncellen zijn echter vaak ongevoelig voor deze methoden vanwege de lage efficiëntie van vectorafgifte door transfectie of transductie. Vervolgens zijn methoden ontwikkeld om sgRNA-Cas9-complexen in vitro te genereren en deze via elektroporatie af te leveren, en deze methoden hebben een hoge efficiëntie bereikt in verschillende celtypen13,14. De resultaten suggereren de mogelijkheid om deze aanpak te gebruiken om genoombewerking uit te voeren in primaire macrofagen.
Hier bieden we een protocol voor het gebruik van sgRNA-Cas9 ribonucleoproteïnecomplexen (RNP's) om genoombewerking uit te voeren in primaire BMDM's. Het bevat stappen om de activering van de immuunsensoren die aanwezig zijn in primaire immuuncellen te verminderen en resulteert in tot 95% bewerking op gerichte loci met minimale toxiciteit. Dit protocol omvat ook workflows om de bewerkingsefficiëntie te evalueren met behulp van routinematige polymerasekettingreactie (PCR) en Sanger-sequencing, gevolgd door in silico-analyse door Tracking of Indels by Decomposition (TIDE)15, een goed gevalideerde online softwaretool.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3T3-MCSF Cell Line</td>
			<td>Gift from Russell Vance</td>
			<td>not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Alt-R Cas9 Electroporation Enhancer</td>
			<td>IDT</td>
			<td>1075915</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampure XP Reagent Beads</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td></td>
		</tr>
		<tr>
			<td>Calf intestinal alkaline phosphatase</td>
			<td>NEB</td>
			<td>M0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>NEB</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS +Ca/Mg (0.9mM CaCl2 and 0.5mM MgCl2)</td>
			<td>Thermo Fisher</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS -Ca/Mg</td>
			<td>Thermo Fisher</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>ExoI</td>
			<td>NEB</td>
			<td>M0293S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FCS)</td>
			<td>Corning</td>
			<td>35-015-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Herculase DNA polymerase &#38; buffer</td>
			<td>Agilent</td>
			<td>600677</td>
			<td></td>
		</tr>
		<tr>
			<td>HiScribe T7 High Yield RNA Synthesis Kit</td>
			<td>NEB</td>
			<td>E2040S</td>
			<td></td>
		</tr>
		<tr>
			<td>LoBind conical tubes 15 mL</td>
			<td>Eppendorf</td>
			<td>30122216</td>
			<td></td>
		</tr>
		<tr>
			<td>LoBind Eppendorf tubes 2 mL</td>
			<td>Eppendorf</td>
			<td>22431102</td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer r2.1</td>
			<td>NEB</td>
			<td>B6002S</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Thermo Fisher</td>
			<td>MPK5000, MPP100, MPS100</td>
			<td></td>
		</tr>
		<tr>
			<td>Neon Transfection System 10 uL Tips</td>
			<td>Thermo Fisher</td>
			<td>MPK1025 or MPK1096</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS + 1mM EDTA</td>
			<td>Lonza</td>
			<td>BE02017F</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Thermo Fisher</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>rCutSmart Buffer for ExoI</td>
			<td>NEB</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ribolock</td>
			<td>Thermo Fisher</td>
			<td>EO0384</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA loading dye</td>
			<td>NEB</td>
			<td>B0363S</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>S. pyogenes Cas9-NLS</td>
			<td>University of California Macro Lab</td>
			<td>not applicable</td>
			<td>Available to non-UC investigators through&#160; https://qb3.berkeley.edu</td>
		</tr>
		<tr>
			<td>S. pyogenes Cas9-NLS, modified 3rd Generation</td>
			<td>IDT</td>
			<td>1081059</td>
			<td></td>
		</tr>
		<tr>
			<td>SAP</td>
			<td>NEB</td>
			<td>M0371S</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-efficiency gene disruption in primary bone marrow-derived macrophages using electroporated cas9-sgrna complexes

Van beenmerg afgeleide macrofagen (BMDM's) van muizen zijn een belangrijk hulpmiddel voor het bestuderen van de complexe biologie van weefselmacrofagen. Als primaire cellen modelleren ze de fysiologie van macrofagen in vivo nauwkeuriger dan onsterfelijke macrofaagcellijnen en kunnen ze worden afgeleid van muizen die al gedefinieerde genetische veranderingen dragen. Het verstoren van de genfunctie in BMDM's blijft echter technisch uitdagend. Hier bieden we een protocol voor efficiënte CRISPR/Cas9-genoombewerking in BMDM's, dat de introductie van kleine inserties en deleties (indels) mogelijk maakt die resulteren in frameshift-mutaties die de genfunctie verstoren. Het protocol beschrijft hoe single-guide RNA's (sgRNA-Cas9) kunnen worden gesynthetiseerd en gezuiverde sgRNA-Cas9 ribonucleoproteïnecomplexen (RNP's) kunnen worden gevormd die kunnen worden afgeleverd door elektroporatie. Het biedt ook een efficiënte methode voor het bewaken van de bewerkingsefficiëntie met behulp van routinematige Sanger-sequencing en een gratis beschikbaar online analyseprogramma. Het protocol kan binnen 1 week worden uitgevoerd en vereist geen plasmideconstructie; Het resulteert doorgaans in een bewerkingsefficiëntie van 85% tot 95%.

Macrophages are innate immune cells that play critical roles in tissue repair and immunity1,2. Immortalized macrophage cell lines, such as mouse RAW 264.7 cells or human THP-1 cells, have several beneficial characteristics, including robust growth and ease of gene disruption by delivering vectors for RNA interference or CRISPR/Cas93,4. However, oncogenic transformation dramatically alters their physiology, which results in the aberrant activation of some pathways and muted responses of others5,6. Primary bone marrow-derived macrophages (BMDMs) more closely recapitulate in vivo macrophage physiology, but remain challenging to genetically manipulate due to the low efficiency of both plasmid transfection and viral transduction in these primary immune cells7,8. Thus, more efficient methods for disrupting gene function are needed.
CRISPR/Cas9 genome editing is a powerful tool for genetic manipulation across a range of biological systems, including mammalian cells9,10,11,12. The Streptococcus pyogenes Cas9 protein efficiently and specifically cleaves double-stranded DNA when complexed with a sequence-specific guide RNA. DNA repair through the non-homologous end joining (NHEJ) of the cleaved DNA results in small insertions or deletions (indels) that create frameshift mutations. In early studies, Cas9 and sgRNAs were delivered through plasmid or lentiviral vectors, which are effective delivery methods for many cell lines9,10. However, primary cells and, in particular, primary immune cells are often refractory to these methods due to the low efficiency of vector delivery by transfection or transduction. Subsequently, methods have been developed to generate sgRNA-Cas9 complexes in vitro and to deliver them via electroporation, and these methods have achieved high efficiency in a variety of cell types13,14. The results have suggested the possibility of using this approach to carry out genome editing in primary macrophages.
Here, we provide a protocol for using sgRNA-Cas9 ribonucleoprotein complexes (RNPs) to carry out genome editing in primary BMDMs. It contains steps to mitigate the activation of the immune sensors present in primary immune cells and results in up to 95% editing at targeted loci with minimal toxicity. This protocol also includes workflows to evaluate editing efficiency using routine polymerase chain reaction (PCR) and Sanger sequencing, followed by in silico analysis by Tracking of Indels by Decomposition (TIDE)15, a well-validated online software tool.

Auteur in de schijnwerpers: Efficiënte CRISPR/Cas9-genoombewerking in van beenmerg afgeleide macrofagen voor nauwkeurige genverstoring

Zeer efficiënte genverstoring in primaire beenmerg-afgeleide macrofagen met behulp van geëlektropoleerde Cas9-sgRNA-complexen

Our laboratory studies the interaction between mycobacterium, tuberculosis and the host immune system, with a focus on the initial interaction between the bacterium and the macrophage. There's a variety of gene-disruption techniques used in macrophages, including RNAi technologies and CRISPR technologies. One of the major challenges is the low efficiency of gene disruption in macrophages.This protocol enables the rapid and high-efficiency genetic manipulation of primary macrophages without the need for cloning and plasmid construction. These methods should be broadly applicable in immunology, enabling researchers to disrupt genes in primary macrophages for a wide array of studies.

De IVT-template is een PCR-product van 127 bp (Figuur 1B). Het IVT-product van volledige lengte is een 98 nt RNA, dat op dezelfde manier migreert als een dubbelstrengs DNA-fragment van 70 bp (Figuur 1C).
Na elektroporatie zouden de cellen >90% levensvatbaar moeten zijn, met een totaal celgetal van >70% van het startcelaantal. De resulterende pool van gemuteerde cellen zou een diverse set indels moeten hebben, beginnend in de buurt van...

Watch this Scientific Journal Video about Author Spotlight: Efficient CRISPR/Cas9 Genome Editing in Bone Marrow-Derived Macrophages for Precise Gene Disruption at JoVE.com

Dit protocol beschrijft de procedure voor genoombewerking in van beenmerg afgeleide macrofagen van muizen met behulp van Cas9-sgRNA ribonucleoproteïnecomplexen die in vitro zijn geassembleerd en door elektroporatie worden afgeleverd.

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the ...

Ons laboratorium bestudeert de interactie tussen mycobacterium, tuberculose en het immuunsysteem van de gastheer, met een focus op de initiële interactie tussen de bacterie en de macrofaag. Er zijn verschillende technieken voor genverstoring die worden gebruikt in macrofagen, waaronder RNAi-technologieën en CRISPR-technologieën. Een van de grootste uitdagingen is de lage efficiëntie van genverstoring in macrofagen.Dit protocol maakt de snelle en zeer efficiënte genetische manipulatie van primaire macrofagen mogelijk zonder de noodzaak van klonen en plasmideconstructie. Deze methoden moeten breed toepasbaar zijn in de immunologie, zodat onderzoekers genen in primaire macrofagen kunnen verstoren voor een breed scala aan studies.

high-efficiency-gene-disruption-primary-bone-marrow-derived

Research

JoVE Journal

Genetics

High-Efficiency Gene Disruption in Primary Bone Marrow-Derived Macrophages Using Electroporated Cas9-sgRNA Complexes

Bone marrow-derived macrophages (BMDMs) from mice are a key tool for studying the complex biology of tissue macrophages. As primary cells, they model the physiology of macrophages in vivo more closely than immortalized macrophage cell lines and can be derived from mice already carrying defined genetic changes. However, disrupting gene function in BMDMs remains technically challenging. Here, we provide a protocol for efficient CRISPR/Cas9 genome editing in BMDMs, which allows for the introduction of small insertions and deletions (indels) that result in frameshift mutations that disrupt gene function. The protocol describes how to synthesize single-guide RNAs (sgRNA-Cas9) and form purified sgRNA-Cas9 ribonucleoprotein complexes (RNPs) that can be delivered by electroporation. It also provides an efficient method for monitoring editing efficiency using routine Sanger sequencing and a freely available online analysis program. The protocol can be performed within 1 week and does not require plasmid construction; it typically results in 85% to 95% editing efficiency.

This protocol describes the procedure for genome editing in mouse bone marrow-derived macrophages using Cas9-sgRNA ribonucleoprotein complexes assembled in vitro and delivered by electroporation.

Preparation for Electroporation and Ribonucleoprotein Delivery for Genome Editing in Mouse Bone Marrow-Derived Macrophages

To begin, set the parameters of the pipette station placed in the laminar flow hood to 1, 900 volts, 20 milliseconds in one pulse. Carefully remove a transfection tube from the package to keep it sterile and fill it with three milliliters of E buffer. To prepare the cells for electroporation, remove the growth medium from the cells and wash them using PBS to remove the serum and divalent cations that promote adhesion.Then add a mixture of PBS and one millimolar EDTA to the cells and incubate at room temperature for about five minutes until the cells begin to detach. Pipette up and down gently to detach the cells and transfer the cells to a low protein binding 15 milliliter tube. Pellet the cells at 500 G for five minutes.After centrifugation quickly remove most of the supernatant, leaving about one milliliter of the supernatant in the tube. Resuspend the cells in the remaining supernatant and transfer it to a low protein binding 1.5 milliliter microfuge tube. Remove a small aliquot of cells to count and pellet the remaining cells by centrifusion at 500 G at room temperature for five minutes.Count the cells during centrifugation. Take 1x sgRNA in 20 millimolar Cas9 solution and warm the tubes to room temperature. After centrifugation, remove all the supernatant from the cell pellet and resuspend the cells in PBS with calcium chloride and magnesium chloride at a concentration of 40 million cells per milliliter.For the sgRNA Cas9 ribonucleoprotein complex assembly, add 2.5 microliters of sgRNA to one microliter of the diluted Cas9 in sterilized PCR tubes and dispense the sgRNA slowly with mixing for about 15 seconds to prevent precipitation. Incubate for five minutes at room temperature to allow the ribonucleoprotein complex formation. To deliver the ribonucleoprotein complex by electroporation, prepare the electroporation tip by depressing the plunger to extend the stem.Then insert the stem into the tip. Resuspend the cells by pipetting up and down. Load the electroporation tip with the cells and withdraw the full 10 microliters volume capacity of the tip.Starting with the negative control sgRNA, transfer 10 microliters of cells in the electroporation tip to the sample tube containing 3.5 microliters of ribonucleoprotein. Pipette up and down three times to mix well, and draw 10 microliters of the mixture back into the tip. Place the tip into the electroporation station, lowering it into the buffer, and push Start on the touchscreen display.After completion, the display will indicate a successful pulse. Remove the pipette from the device and place the cells in a dry well of the uncoated 12 well plate. Rinse the tip by pipetting up and down twice in 15 milliliters of PBS with calcium chloride and magnesium chloride.Set aside the pipette with the tip still attached. Immediately add one milliliter of the antibiotic free medium aliquoted earlier to the cells and gently shake the plate to mix. The representative Sanger sequencing chromatogram of the Rosa 26 locus from the bone marrow derived macrophages electroporated with a controlled non-targeting sgRNA Cas9 ribonucleoprotein, Rosa 26 specific sgRNA, and Src and Cblb genes in the edited macrophages are shown here.The analysis of the targeted genes by PCR and Sanger sequencing shows multiple nucleotides at each position downstream of the Cas9 cleavage site. These results validate the attainment of a high editing efficiency for multiple targeted genes.