Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

Chu, Van Trung; Graf, Robin; Wirtz, Tristan; Weber, Timm; Favret, Jérémy; Li, Xun; Petsch, Kerstin; Tran, Ngoc Tung; Sieweke, Michael H.; Berek, Claudia; Kühn, Ralf; Rajewsky, Klaus

doi:10.1073/pnas.1613884113

Cited by 117 publications

(140 citation statements)

References 27 publications

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“…A second reactivation step with half the concentration of mAb was necessary for efficient editing. Remarkably, despite the notion that DNA-based CRISPR/Cas editing does not work in T cells, the gene ablation efficiencies are equal to alternative protocols using in vitro transcribed sgRNA/Cas9 mRNA or RNPs (2), (3) and even to the protocol using viral transduction of transgenic Cas9 expressing T cells (6). Next we combined the plasmids targeting CD90.2 and CD45.2.…”

Section: Resultsmentioning

confidence: 99%

“…While previous efforts to establish CRISPR/Cas9-mediated gene editing in primary T cells have mainly focused on human T cells (1), (2), (3), (4) to our knowledge only two reports describe successful CRISPR/Cas9 gene editing in primary murine T cells (5), (6). Both approaches depend on mice expressing transgenic Cas9 and either a second transgenic construct to express the gRNA (5), or viral transduction of T cells followed by antibiotic selection (6).…”

Section: Introductionmentioning

confidence: 99%

“…Both approaches depend on mice expressing transgenic Cas9 and either a second transgenic construct to express the gRNA (5), or viral transduction of T cells followed by antibiotic selection (6). As a result, although these approaches constitute significant advances, they still require breeding of a constitutive Cas9 transgene, which may be genotoxic and might lead to immunologic rejection after adoptive transfer of transgenic cells.…”

Section: Introductionmentioning

confidence: 99%

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Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

Kornete

Marone

Jeker

2018

The Journal of Immunology

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Adoptive cell transfer (ACT) is an important approach for basic research and emerges as an effective treatment for various diseases including infections and blood cancers. Direct genetic manipulation of primary immune cells opens up unprecedented research opportunities and could be applied to enhance cellular therapeutic products. Here, we report highly efficient genome engineering in primary murine T cells using a plasmid-based RNA-guided CRISPR system. We developed a straightforward approach to ablate genes in up to 90% of cells and to introduce precisely targeted single nucleotide polymorphisms (SNP) in up to 25% of the transfected primary T cells. We used gene editing-mediated allele switching to quantify homology directed repair (HDR), systematically optimize experimental parameters and map a native B cell epitope in primary T cells. Allele switching of a surrogate cell surface marker can be used to enrich cells with successful simultaneous editing of a second gene of interest. Finally, we applied the approach to correct two disease-causing mutations in the Foxp3 gene. Both repairing the cause of the scurfy syndrome, a 2bp insertion in Foxp3, and repairing the clinically relevant Foxp3K276X mutation restored Foxp3 expression in primary T cells.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

Kornete

Marone

Jeker

2018

The Journal of Immunology

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“…The transfection of miR122a into the Caco-2 cells led to a decreased expression of EGFR only in the protein level determined by Western blot, which was inhibited at the posttranscript level, whereas the mRNA of EGFR was unaffected, as a regulatory mechanism of microRNA [1,15,28]. The cotranscription of EGFR with miR-122a could cover the effects of miR-122a on the increased expression level of zonulin possibly because the increased EGFR maintained the molecular regulatory inhibition on the expression of zonulin.…”

Section: Discussionmentioning

confidence: 98%

“…MiR-122a intestinal transgenic mice (mir-122a-TG) were generated by Cyagen Biosciences Inc. (Guangzhou, China) using a villin promoter and background mice of C57BL/6, as described [15]. Mice were bred and kept at the Sun Yat-Sen University Laboratory Animal Center (Guangzhou, China).…”

Section: Building and Identifying Mir-122a Transgenic Micementioning

confidence: 99%

MicroRNA-122a Regulates Zonulin by Targeting EGFR in Intestinal Epithelial Dysfunction

Zhang

Tian

Ping

et al. 2017

Cell Physiol Biochem

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Background/Aims: This study aimed to investigate the role of microRNA (miR)-122a in regulating zonulin during the modulation of intestinal barrier. Methods: Zonulin proteins and their target gene expression were analyzed in miR-122a-overexpressing cell lines and in the target gene of epidermal growth factor receptor (EGFR). An mmu-miR-122a intestinal epithelial conditional transgenic (miR-122a-TG) mouse model was established to investigate EGFR and zonulin expression. MiR-122a was also detected in the clinical specimens of inflammatory bowel disease. Results: EGFR was identified as a target gene of miR-122a. The expression level of miR-122a was positively correlated with that of zonulin. The expression level of zonulin was significantly increased, whereas the expression level of EGFR was significantly decreased in the miR-122a-TG mice and in the corresponding primary epithelial culture (P < 0.05). These results were consistent with the data of the clinical specimens. Conclusions: miR-122a could be a positive factor of zonulin by targeting EGFR, which increased the intestinal epithelial permeability in vivo and in vitro.

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CRISPR/Cas9 Genome Editing Using Gold‐Nanoparticle‐Mediated Laserporation

et al. 2018

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Engineered nucleases hold large potential for future gene therapy applications. An obstacle hampering their applications are delivery methods bearing efficiency, throughput, and viability of target cells. How this limitation can be overcome via gold‐nanoparticle‐mediated (GNOME) laserporation is demonstrated. It employs a picosecond laser setup and 200 nm gold nanoparticles, and its full capacity with CRISPR/Cas9 delivery is demonstrated. 70 kDa dextrans are utilized to probe delivery in adherent SC1 cells. Afterward, GNOME laserporation is used for transfection of crRNA:tracrRNA targeting the mouse CCR7 (mCCR7) into SpCas9 (Streptococcus pyogenes Cas9) and mCCR7 co‐expressing SC1 cells. Finally, ribonucleoprotein particles consisting of mCCR7 crRNA:tracrRNA and SpCas9 endonuclease are transfected into SC1 cells not expressing SpCas9. Gene knockout efficiencies of up to 65% are detected in the GNOME laserporated cells. To validate the simplicity of the approach, the same treatment parameters are used to successfully knock out CXCR3 in 25% of GNOME laserporated activated mouse CD8+ T cells. In conclusion, this is the first demonstration of the unique combination of nanotechnology and laser irradiation for gene editing via engineered nucleases.

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Efficient CRISPR-mediated mutagenesis in primary immune cells using CrispRGold and a C57BL/6 Cas9 transgenic mouse line

Cited by 117 publications

References 27 publications

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

Highly Efficient and Versatile Plasmid-Based Gene Editing in Primary T Cells

MicroRNA-122a Regulates Zonulin by Targeting EGFR in Intestinal Epithelial Dysfunction

CRISPR/Cas9 Genome Editing Using Gold‐Nanoparticle‐Mediated Laserporation

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