Name: 저자 스포트라이트: 정확한 유전자 파괴를 위한 골수 유래 대식세포의 효율적인 CRISPR/Cas9 게놈 편집
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

이 연구는 NIH 보조금 5R01AI144149의 지원을 받았습니다. 회로도는 BioRender로 제작되었습니다.

1. sgRNA 디자인
NOTE: 이 단계에서는 sgRNA의 표적 염기서열 선택 및 설계에 대해 설명합니다. 첫 번째 큰 코딩 엑손(big coding exon)에 있는 가이드를 설계하여 번역된 단백질이 열린 판독 프레임 초기에 파괴되도록 하는 것이 도움이 됩니다. 또한 동일한 엑손 내에 있는 표적 염기서열을 선택하는 것이 도움이 되는데, 이는 편집 효율 분석을 간소화하기 때문입니다(6단계)....

A Guide to Precise Gene Disruption in Primary Bone Marrow-Derived macrophages(원발성 골수 유래 대식세포의 정확한 유전자 파괴에 대한 가이드)

<ol>
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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>

전기천공된 Cas9-sgRNA 복합체를 사용한 게놈 편집은 BMDM에서 유전자 기능을 효과적으로 방해할 수 있습니다. 편집 효율은 표적 염기서열과 유전자에 따라 다릅니다. 일반적으로 4-5개의 sgRNA를 스크리닝하여 활성이 높은 sgRNA를 식별합니다. 일부 유전자좌는 편집 효율이 낮은데, 이는 염색질 구조 때문일 가능성이 큽니다. 이러한 경우 편집 효율성을 높이기 위해 몇 가지 수정을 수행할 수 있습니다. ?...

대식세포는 조직 복구와 면역에 중요한 역할을 하는 선천성 면역 세포입니다 1,2. 마우스 RAW 264.7 세포 또는 인간 THP-1 세포와 같은 불멸화된 대식세포 세포주는 RNA 간섭 또는 CRISPR/Cas9 3,4에 대한 벡터를 전달하여 강력한 성장과 유전자 파괴 용이성을 포함하여 여러 가지 유익한 특성을 가지고 있습니다. 그러나 종양 변형은 생리학을 극적으로 변화시켜 일부 경로의 비정상적인 활성화와 다른 경로의 음소거 반응을 초래합니다 5,6. 일차 골수 유래 대식세포(BMDM)는 생체 내 대식세포 생리학을 보다 밀접하게 재현하지만, 이러한 일차 면역 세포에서 플라스미드 형질주입 및 바이러스 형질주입의 효율성이 낮기 때문에 유전적으로 조작하는 것이 여전히 어렵습니다 7,8. 따라서 유전자 기능을 교란하기 위한 보다 효율적인 방법이 필요하다.
CRISPR/Cas9 게놈 편집은 포유류 세포 9,10,11,12를 포함한 다양한 생물학적 시스템에서 유전자 조작을 위한 강력한 도구입니다. Streptococcus pyogenes Cas9 단백질은 염기서열 특이적 가이드 RNA와 복합체될 때 이중 가닥 DNA를 효율적이고 특이적으로 절단합니다. 절단된 DNA의 NHEJ(non-homologous end joining)를 통한 DNA 복구는 프레임시프트 돌연변이를 생성하는 작은 삽입 또는 결실(indels)을 초래합니다. 초기 연구에서 Cas9 및 sgRNA는 플라스미드 또는 렌티바이러스 벡터를 통해 전달되었으며, 이는 많은 세포주에 대한 효과적인 전달 방법입니다 9,10. 그러나, 일차 세포, 특히 일차 면역 세포는 형질주입 또는 형질도입에 의한 벡터 전달의 효율이 낮기 때문에 종종 이러한 방법에 불응한다. 그 후, 시험관 내에서 sgRNA-Cas9 복합체를 생성하고 전기천공법을 통해 전달하는 방법이 개발되었으며, 이러한 방법은 다양한 세포 유형에서 높은 효율을 달성했습니다13,14. 결과는 1차 대식세포에서 게놈 편집을 수행하기 위해 이 접근법을 사용할 가능성을 시사했습니다.
여기서는 sgRNA-Cas9 리보핵단백질 복합체(RNP)를 사용하여 1차 BMDM에서 게놈 편집을 수행하기 위한 프로토콜을 제공합니다. 여기에는 일차 면역 세포에 존재하는 면역 센서의 활성화를 완화하는 단계가 포함되어 있으며 최소한의 독성으로 표적 유전자좌에서 최대 95%까지 편집됩니다. 이 프로토콜에는 일상적인 중합효소연쇄반응(PCR) 및 Sanger 염기서열분석을 사용하여 편집 효율성을 평가하는 워크플로우와 검증된 온라인 소프트웨어 도구인 TIDE(Tracking of Indels by Decomposition)15를 통한 인실리코(in silico) 분석도 포함됩니다.

<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

생쥐의 골수 유래 대식세포(BMDM)는 조직 대식세포의 복잡한 생물학을 연구하기 위한 핵심 도구입니다. 일차 세포로서 생체 내 대식세포의 생리학을 불멸화된 대식세포 세포주보다 더 밀접하게 모델링하며 이미 정의된 유전적 변화를 가지고 있는 마우스에서 파생될 수 있습니다. 그러나 BMDM에서 유전자 기능을 방해하는 것은 기술적으로 여전히 어려운 과제입니다. 여기서는 BMDM에서 효율적인 CRISPR/Cas9 게놈 편집을 위한 프로토콜을 제공하며, 이를 통해 유전자 기능을 방해하는 프레임시프트 돌연변이를 초래하는 작은 삽입 및 결실(indel)을 도입할 수 있습니다. 이 프로토콜은 단일 가이드 RNA(sgRNA-Cas9)를 합성하고 전기천공법으로 전달할 수 있는 정제된 sgRNA-Cas9 리보핵단백질 복합체(RNP)를 형성하는 방법을 설명합니다. 또한 일상적인 Sanger 염기서열분석과 무료로 제공되는 온라인 분석 프로그램을 사용하여 편집 효율성을 모니터링하는 효율적인 방법을 제공합니다. 프로토콜은 1주일 이내에 수행할 수 있으며 플라스미드 구축이 필요하지 않습니다. 일반적으로 85%에서 95%의 편집 효율성을 얻을 수 있습니다.

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.

저자 스포트라이트: 정확한 유전자 파괴를 위한 골수 유래 대식세포의 효율적인 CRISPR/Cas9 게놈 편집

전기천공된 Cas9-sgRNA 복합체를 사용한 일차 골수 유래 대식세포의 고효율 유전자 파괴

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 템플릿은 127bp PCR 산물입니다(그림 1B). 전장 IVT 산물은 98 nt RNA로, 70 bp 이중 가닥 DNA 단편과 유사하게 이동합니다(그림 1C).
electroporation 후에, 세포는 시작 세포 수의 >70%의 총 세포 조사와 더불어 >90% 생존가능해야 합니다. 돌연변이 세포의 결과 풀은 Cas9 절단 부위 근처에서 시작하여 다양한 indels 세트를 가져야 합니다. PCR 및 Sanger...

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

이 프로토콜은 시험관내에서 조립되고 전기천공법으로 전달되는 Cas9-sgRNA 리보핵단백질 복합체를 사용하여 마우스 골수 유래 대식세포에서 게놈 편집 절차를 설명합니다.

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 ...

우리 실험실은 박테리아와 대식세포 사이의 초기 상호 작용에 중점을 두고 마이코박테리움, 결핵 및 숙주 면역 체계 간의 상호 작용을 연구합니다. RNAi 기술 및 CRISPR 기술을 포함하여 대식세포에 사용되는 다양한 유전자 파괴 기술이 있습니다. 주요 과제 중 하나는 대식세포에서 유전자 파괴의 효율성이 낮다는 것입니다.이 프로토콜은 클로닝 및 플라스미드 구축 없이 1차 대식세포의 빠르고 효율적인 유전자 조작을 가능하게 합니다. 이러한 방법은 면역학에 광범위하게 적용되어야 하며, 연구자들은 다양한 연구를 위해 일차 대식세포의 유전자를 파괴할 수 있습니다.

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.

연구

유전학

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.