Name: Forfatter Spotlight: Effektiv CRISPR/Cas9-genomredigering i benmargsavledede makrofager for presis genforstyrrelse
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

Dette arbeidet ble finansiert av NIH-stipendet 5R01AI144149. De skjematiske figurene ble laget med BioRender.

1. sgRNA-design
MERK: Dette trinnet beskriver valg av målsekvenser og utforming av sgRNAene. Det er nyttig å designe guider som er i det første store kodingseksonet, slik at ethvert oversatt protein blir forstyrret tidlig i den åpne leserammen. Det er også nyttig å velge målsekvenser som ligger innenfor samme ekson, da dette vil effektivisere analysen av redigeringseffektiviteten (trinn 6). Eksemplene på genomredigering som følger med denne protokollen, brukte sgRNA ret...

En veiledning til presis genforstyrrelse i primære benmargsavledede makrofager

<ol>
	<li>Murray, P. J., Wynn, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+and+pathogenic+functions+of+macrophage+subsets.">Protective and pathogenic functions of macrophage subsets.</a> Nature Reviews Immunology. 11 (11), 723-737 (2011).</li><li>Wynn, T. A., Chawla, A., Pollard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage+biology+in+development,+homeostasis,+and+disease.">Macrophage biology in development, homeostasis, and disease.</a> Nature. 496 (7446), 445-455 (2013).</li><li>Paddison, P. J., Caudy, A. A., Hannon, G. 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>

Genomredigering ved hjelp av elektroporerte Cas9-sgRNA-komplekser tillater effektiv forstyrrelse av genfunksjonen i BMDM. Redigeringseffektiviteten varierer etter målsekvens og gen. Vanligvis blir fire til fem sgRNA generelt screenet for å identifisere en som er svært aktiv. Noen loci har lavere redigeringseffektivitet, mest sannsynlig på grunn av kromatinstrukturen. I disse tilfellene kan flere modifikasjoner gjøres for å øke redigeringseffektiviteten. Samlevering av to aktive sgRNA til samme ekson resulterer i f...

Makrofager er medfødte immunceller som spiller kritiske roller i vevsreparasjon og immunitet 1,2. Immortaliserte makrofagcellelinjer, som mus RAW 264.7-celler eller humane THP-1-celler, har flere fordelaktige egenskaper, inkludert robust vekst og enkel genforstyrrelse ved å levere vektorer for RNA-interferens eller CRISPR / Cas9 3,4. Imidlertid endrer onkogen transformasjon dramatisk deres fysiologi, noe som resulterer i avvikende aktivering av noen veier og dempede responser fra andre 5,6. Primære benmargsavledede makrofager (BMDM) rekapituleres nærmere in vivo makrofagfysiologi, men er fortsatt utfordrende å genetisk manipulere på grunn av den lave effektiviteten av både plasmidtransfeksjon og viral transduksjon i disse primære immuncellene 7,8. Dermed er det behov for mer effektive metoder for å forstyrre genfunksjonen.
CRISPR/Cas9 genomredigering er et kraftig verktøy for genetisk manipulering på tvers av en rekke biologiske systemer, inkludert pattedyrceller 9,10,11,12. Streptococcus pyogenes Cas9-proteinet spalter effektivt og spesifikt dobbeltstrenget DNA når det kompleksiseres med et sekvensspesifikt guide-RNA. DNA-reparasjon gjennom den ikke-homologe endesammenføyningen (NHEJ) av det spaltede DNA-et resulterer i små innsettinger eller delesjoner (indels) som skaper frameshift-mutasjoner. I tidlige studier ble Cas9 og sgRNA levert gjennom plasmid- eller lentivirale vektorer, som er effektive leveringsmetoder for mange cellelinjer 9,10. Imidlertid er primære celler og spesielt primære immunceller ofte ildfaste mot disse metodene på grunn av den lave effektiviteten av vektorlevering ved transfeksjon eller transduksjon. Deretter har metoder blitt utviklet for å generere sgRNA-Cas9-komplekser in vitro og å levere dem via elektroporering, og disse metodene har oppnådd høy effektivitet i en rekke celletyper13,14. Resultatene har antydet muligheten for å bruke denne tilnærmingen til å utføre genomredigering i primære makrofager.
Her gir vi en protokoll for bruk av sgRNA-Cas9 ribonukleoproteinkomplekser (RNP) for å utføre genomredigering i primære BMDM. Den inneholder trinn for å redusere aktiveringen av immunsensorene som er tilstede i primære immunceller og resulterer i opptil 95% redigering på målrettede steder med minimal toksisitet. Denne protokollen inkluderer også arbeidsflyter for å evaluere redigeringseffektivitet ved hjelp av rutinemessig polymerasekjedereaksjon (PCR) og Sanger-sekvensering, etterfulgt av i silikoanalyse av Tracking of Indels by Decomposition (TIDE)15, et godt validert online programvareverktøy.

<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

Benmargsavledede makrofager (BMDM) fra mus er et sentralt verktøy for å studere den komplekse biologien til vevsmakrofager. Som primære celler modellerer de fysiologien til makrofager in vivo nærmere enn immortaliserte makrofagcellelinjer og kan stamme fra mus som allerede bærer definerte genetiske endringer. Imidlertid er forstyrrende genfunksjon i BMDM fortsatt teknisk utfordrende. Her gir vi en protokoll for effektiv CRISPR/Cas9-genomredigering i BMDM, som muliggjør innføring av små innsettinger og delesjoner (indels) som resulterer i frameshift-mutasjoner som forstyrrer genfunksjonen. Protokollen beskriver hvordan man syntetiserer enkeltguidede RNA (sgRNA-Cas9) og danner rensede sgRNA-Cas9 ribonukleoproteinkomplekser (RNP) som kan leveres ved elektroporering. Det gir også en effektiv metode for å overvåke redigeringseffektivitet ved hjelp av rutinemessig Sanger-sekvensering og et fritt tilgjengelig online analyseprogram. Protokollen kan utføres innen 1 uke og krever ikke plasmidkonstruksjon; Det resulterer vanligvis i 85% til 95% redigeringseffektivitet.

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.

Forfatter Spotlight: Effektiv CRISPR/Cas9-genomredigering i benmargsavledede makrofager for presis genforstyrrelse

Høyeffektiv genforstyrrelse i primære benmargsavledede makrofager ved bruk av elektroporerte Cas9-sgRNA-komplekser

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.

IVT-malen er et 127 bp PCR-produkt (figur 1B). IVT-produktet i full lengde er et 98 nt RNA, som migrerer på samme måte som et 70 bp dobbeltstrenget DNA-fragment (figur 1C).
Etter elektroporering skal cellene være >90% levedyktige, med et totalt celletall på >70% av startcellenummeret. Det resulterende bassenget av mutante celler bør ha et mangfoldig sett med indels, som starter nær Cas9-spaltningsstedet. Analysen av de målrette...

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

Denne protokollen beskriver prosedyren for genomredigering i musbenmargsavledede makrofager ved bruk av Cas9-sgRNA ribonukleoproteinkomplekser samlet in vitro og levert ved elektroporering.

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 ...

Vårt laboratorium studerer samspillet mellom mykobakterie, tuberkulose og vertsimmunsystemet, med fokus på det første samspillet mellom bakterien og makrofagen. Det finnes en rekke genforstyrrelsesteknikker som brukes i makrofager, inkludert RNAi-teknologier og CRISPR-teknologier. En av de store utfordringene er den lave effektiviteten av genforstyrrelser i makrofager.Denne protokollen muliggjør rask og høyeffektiv genetisk manipulering av primære makrofager uten behov for kloning og plasmidkonstruksjon. Disse metodene bør være bredt anvendelige i immunologi, slik at forskere kan forstyrre gener i primære makrofager for et bredt spekter av studier.

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

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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.