Name: Forfatter Spotlight: Effektiv CRISPR/Cas9-genomredigering i knoglemarvsafledte makrofager for præcis 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 arbejde blev finansieret af NIH-tilskuddet 5R01AI144149. De skematiske figurer blev oprettet med BioRender.

1. sgRNA-design
BEMÆRK: Dette trin beskriver valg af målsekvenser og design af sgRNA'erne. Det er nyttigt at designe guider, der er i den første store kodende exon, så ethvert oversat protein forstyrres tidligt i den åbne læseramme. Det er også nyttigt at vælge målsekvenser, der ligger inden for samme exon, da dette vil strømline analysen af redigeringseffektiviteten (trin 6). Eksemplerne på genomredigering, der leveres med denne protokol, brugte sgRNA'er rettet mod d...

En guide til præcis genforstyrrelse i primære knoglemarvsafledte 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 hjælp af elektroporerede Cas9-sgRNA-komplekser muliggør effektiv forstyrrelse af genfunktionen i BMDM'er. Redigeringseffektiviteten varierer efter målsekvens og gen. Typisk screenes fire til fem sgRNA'er generelt for at identificere en, der er meget aktiv. Nogle loci har lavere redigeringseffektivitet, sandsynligvis på grund af kromatinstrukturen. I disse tilfælde kan der foretages flere ændringer for at øge redigeringseffektiviteten. Samtidig levering af to aktive sgRNA'er til samme exon resul...

Makrofager er medfødte immunceller, der spiller kritiske roller i vævsreparation og immunitet 1,2. Udødeliggjorte makrofagcellelinjer, såsom mus RAW 264.7-celler eller humane THP-1-celler, har flere gavnlige egenskaber, herunder robust vækst og let genforstyrrelse ved at levere vektorer til RNA-interferens eller CRISPR / Cas9 3,4. Imidlertid ændrer onkogen transformation dramatisk deres fysiologi, hvilket resulterer i den afvigende aktivering af nogle veje og dæmpede reaktioner fra andre 5,6. Primære knoglemarvsafledte makrofager (BMDM'er) rekapitulerer tættere in vivo makrofagfysiologi, men forbliver udfordrende at genetisk manipulere på grund af den lave effektivitet af både plasmidtransfektion og viral transduktion i disse primære immunceller 7,8. Der er således behov for mere effektive metoder til at forstyrre genfunktionen.
CRISPR/Cas9 genomredigering er et kraftfuldt værktøj til genetisk manipulation på tværs af en række biologiske systemer, herunder pattedyrceller 9,10,11,12. Streptococcus pyogenes Cas9-proteinet spalter effektivt og specifikt dobbeltstrenget DNA, når det er kompleksbundet med et sekvensspecifikt guide-RNA. DNA-reparation gennem den ikke-homologe endesammenføjning (NHEJ) af det spaltede DNA resulterer i små indsættelser eller deletioner (indels), der skaber frameshift-mutationer. I tidlige undersøgelser blev Cas9 og sgRNA'er leveret gennem plasmid- eller lentivirale vektorer, som er effektive leveringsmetoder for mange cellelinjer 9,10. Imidlertid er primære celler og især primære immunceller ofte ildfaste over for disse metoder på grund af den lave effektivitet af vektorlevering ved transfektion eller transduktion. Efterfølgende er der udviklet metoder til at generere sgRNA-Cas9-komplekser in vitro og levere dem via elektroporation, og disse metoder har opnået høj effektivitet i en række celletyper13,14. Resultaterne har antydet muligheden for at bruge denne tilgang til at udføre genomredigering i primære makrofager.
Her leverer vi en protokol til brug af sgRNA-Cas9 ribonukleoproteinkomplekser (RNP'er) til at udføre genomredigering i primære BMDM'er. Den indeholder trin til at afbøde aktiveringen af immunsensorerne, der findes i primære immunceller og resulterer i op til 95% redigering på målrettede loci med minimal toksicitet. Denne protokol omfatter også arbejdsgange til evaluering af redigeringseffektivitet ved hjælp af rutinemæssig polymerasekædereaktion (PCR) og Sanger-sekventering efterfulgt af in silico-analyse ved sporing af Indels by Decomposition (TIDE)15, et velvalideret online softwareværktøj.

<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

Knoglemarvsafledte makrofager (BMDM'er) fra mus er et vigtigt redskab til at studere den komplekse biologi af vævsmakrofager. Som primære celler modellerer de fysiologien af makrofager in vivo tættere end udødeliggjorte makrofagcellelinjer og kan udledes af mus, der allerede bærer definerede genetiske ændringer. Imidlertid er forstyrrelse af genfunktionen i BMDM'er fortsat teknisk udfordrende. Her leverer vi en protokol til effektiv CRISPR/Cas9-genomredigering i BMDM'er, som giver mulighed for introduktion af små insertioner og deletioner (indels), der resulterer i frameshift-mutationer, der forstyrrer genfunktionen. Protokollen beskriver, hvordan man syntetiserer enkeltguide-RNA'er (sgRNA-Cas9) og danner oprensede sgRNA-Cas9-ribonukleoproteinkomplekser (RNP'er), der kan leveres ved elektroporation. Det giver også en effektiv metode til overvågning af redigeringseffektivitet ved hjælp af rutinemæssig Sanger-sekventering og et frit tilgængeligt online analyseprogram. Protokollen kan udføres inden for 1 uge og kræver ikke plasmidkonstruktion; Det resulterer typisk 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 knoglemarvsafledte makrofager for præcis genforstyrrelse

Højeffektiv genforstyrrelse i primære knoglemarvafledte makrofager ved anvendelse af elektroporerede 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-skabelonen er et 127 bp PCR-produkt (figur 1B). IVT-produktet i fuld længde er et 98 nt RNA, som migrerer på samme måde som et 70 bp dobbeltstrenget DNA-fragment (figur 1C).
Efter elektroporation skal cellerne være >90% levedygtige med et samlet celletal på >70% af startcellenummeret. Den resulterende pulje af mutantceller skal have et forskelligt sæt indels, der starter nær Cas9-spaltningsstedet. Analysen af de målrettede...

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 protokol beskriver proceduren for genomredigering i museknoglemarvsafledte makrofager ved anvendelse af Cas9-sgRNA ribonukleoproteinkomplekser samlet in vitro og leveret ved elektroporation.

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 ...

Vores laboratorium undersøger samspillet mellem mycobacterium, tuberkulose og værtsimmunsystemet med fokus på den indledende interaktion mellem bakterien og makrofagen. Der er en række genforstyrrelsesteknikker, der anvendes i makrofager, herunder RNAi-teknologier og CRISPR-teknologier. En af de store udfordringer er den lave effektivitet af genforstyrrelser i makrofager.Denne protokol muliggør hurtig og højeffektiv genetisk manipulation af primære makrofager uden behov for kloning og plasmidkonstruktion. Disse metoder bør være bredt anvendelige inden for immunologi, hvilket gør det muligt for forskere at forstyrre gener i primære makrofager til en bred vifte af undersøgelser.

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.