Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Nguyen, David N.; Roth, Theodore L.; Li, P. Jonathan; Chen, Peixin Amy; Apathy, Ryan; Mamedov, Murad R.; Vo, Linda T.; Tobin, Victoria; Goodman, Daniel B.; Shifrut, Eric; Bluestone, Jeffrey A.; Puck, Jennifer M.; Szoka, Francis C.; Marson, Alexander

doi:10.1038/s41587-019-0325-6

Cited by 213 publications

(255 citation statements)

References 17 publications

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“…Building on previous work using CRISPR-Cas9 to render primary human immune cells genetically tractable (Hultquist et al, 2019;Hung et al, 2017;Nguyen et al, 2019;Schumann et al, 2015), we have developed a robust, flexible, and high-throughputcompatible platform for the genetic manipulation of primary human myeloid cells. We have optimized cell isolation, culture and CRISPR-Cas9 RNP nucleofection conditions to allow for rapid and inexpensive generation of isogenic modified cells that can then be differentiated and flexibly incorporated into downstream biochemical and phenotypic assays.…”

Section: Discussionmentioning

confidence: 99%

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Hiatt¹,

Cavero

McGregor

et al. 2020

Preprint

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Genome engineering of primary human cells with CRISPR-Cas9 has revolutionized experimental and therapeutic approaches to cell biology, but human myeloidlineage cells have remained largely genetically intractable. We present a method for delivery of CRISPR-Cas9 ribonucleoprotein (RNP) complexes by nucleofection directly into CD14+ human monocytes purified from peripheral blood, leading to high rates of precise gene knockout. These cells can be efficiently differentiated into monocyte-derived macrophages or dendritic cells. This process yields genetically-edited cells that retain critical markers of both myeloid differentiation and phagocytic function. Genetic ablation of the restriction factor SAMHD1 increased HIV-1 infection more than fifty-fold, demonstrating the power of this system for genotype-phenotype interrogation. This fast, flexible and scalable platform can be used for genetic studies of human myeloid cells in immune signaling, inflammation, cancer immunology, host-pathogen interactions, and beyond, and could facilitate development of novel myeloid cellular therapies.

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Section: Discussionmentioning

confidence: 99%

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Hiatt¹,

Cavero

McGregor

et al. 2020

Preprint

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“…It has been proposed that CD8+ T cells that occupy these differentiation states would provide a rapid response to nearby reactivated virus because they are localized closer to the sites of HIV reservoir persistence within lymphoid tissues (and, in particular, B cell follicles; (68)(69)(70)). Indeed, studies have suggested that lymphoid tissue from humans and non-human primates that naturally control retroviral infection is enriched for CD8+ T cells that express the TRMassociated protein, CD69 (71,86), as well as CD8+ T cells that express the Tfc-associated chemokine receptor, CXCR5 (72,73). Studies in mice suggest that TCF-1 may directly promote the generation of Tfc (38) but inhibit the formation of TRMs (74).…”

Section: Discussionmentioning

confidence: 99%

TCF-1 regulates the stem-like memory potential of HIV-specific CD8+ T cells in elite controllers

Rutishauser

Deguit

Hiatt

et al. 2020

Preprint

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Although many HIV cure strategies seek to expand HIV-specific CD8+ T cells to control the virus, all are likely to fail if cellular exhaustion is not prevented. A loss in stem-like memory properties (i.e., the ability to proliferate and generate secondary effector cells) is a key feature of exhaustion; little is known, however, about how these properties are regulated in human virus-specific CD8+ T cells. We found that virus-specific CD8+ T cells from humans and non-human primates naturally controlling HIV/SIV infection express more of the transcription factor, TCF-1, than noncontrollers. HIV-specific CD8+ T cell TCF-1 expression correlated with memory marker expression and proliferative capacity and declined with antigenic stimulation. CRISPR-Cas9 editing of TCF-1 in human primary T cells demonstrated a direct role in regulating expansion capacity. Collectively, these data suggest that TCF-1 controls the stem-like memory properties of HIV-specific CD8+ T cells and provides a rationale for enhancing this pathway in T cell-based therapeutic strategies for HIV. [Main Text:]

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“…Our data revealed that electroporation may induce numerous off-target effects on the NK cell repertoire, skew NK cell cytokine production profiles, and deleteriously impact NK cell killing capacity. In these studies, we pre-treated the electroporated NK cells with IL-2, as NK cell activation appears to be required for post-electroporation viability 35–39 . Interestingly, the phenotypic changes induced by electroporation were not congruent with the alterations expected to be induced by IL-2 treatment alone.…”

Section: Discussionmentioning

confidence: 99%

“…The most successful non-viral delivery method for primary NK cells is currently electroporation 8,34 . However, electroporated NK cells generally require cytokine stimulation or expansion on genetically-modified feeder cell lines for adequate transfection efficiency and viability post-electroporation 35–39 , thereby impeding the study of NK biology by altering cellular physiology and phenotype 40 . We therefore sought to develop and optimize an efficacious and bio-orthogonal transfection strategy for primary NK cells.…”

Section: Introductionmentioning

confidence: 99%

Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

Wilk

Benner

Vergara

et al. 2020

Preprint

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22 Natural killer (NK) cells are capable of rapid and robust cytotoxicity, making them excellent tools 23 for immunotherapy. However, their recalcitrance to standard transfection techniques has limited 24 both mechanistic studies and clinical applications. Current approaches for NK cell manipulation 25 rely on viral transduction or methods requiring NK cell activation, which can alter NK cell 26 function. Here, we report that non-viral Charge-Altering Releasable Transporters (CARTs) 27 efficiently transfect primary human NK cells with mRNA without relying on NK cell activation. 28 Compared to electroporation, CARTs transfect NK cells two orders of magnitude more 29 efficiently, better preserve cell viability, and cause minimal reconfiguration of NK cell phenotype 30 and function. Finally, we use CARTs to generate highly cytotoxic primary human chimeric 31 antigen receptor NK cells, indicating potential therapeutic utility of this technique. To our 32 knowledge, CARTs represent the first efficacious transfection technique for resting primary NK 33 cells that preserves NK cell phenotype, and can drive new biological discoveries and clinical 34 applications of this understudied lymphocyte subset. 35 36 37 38 Natural killer (NK) cells are a group of innate lymphocytes that coordinate and execute 39 the rapid elimination of neoplastic and virus-infected cells 1,2 . Upon activation through a 40 combinatorial array of germline-encoded inhibitory and activating receptors, NK cells can 41 directly kill their targets via targeted release of perforin-and granzyme-containing granules, as 42 well as coordinate the downstream immune response by secreting pro-inflammatory cytokines 43 like IFNγ and TNFα 3,4 . The rapid and robust cytotoxicity of NK cells makes them excellent 44 assets for antiviral and anticancer immunotherapy, an enthusiasm most recently bolstered by 45 the use of chimeric antigen receptor (CAR) NK cells in treating CD19 + lymphoid cancers with 46 high efficacy and low toxicity 5-13 . Despite their clinical promise, several fundamental facets of 47 human NK cell biology are poorly understood. For example, the molecular underpinnings of 48 human NK cell memory 14-16 and the precise roles of various NK cell receptors in target cell 49 recognition remain unknown. 50 NK cells are notoriously difficult to manipulate, presenting challenges both for a deeper 51 understanding of fundamental NK cell biology and for the use of NK cells in clinical 52 applications 17-21 . Most clinical trials involving genetically-modified NK cells utilize viral 53 transduction (NCT02944162, NCT02892695, NCT03056339, NCT03579927) 5,22 . While viral 54 transduction enables stable transgene expression, it is costly, laborious, and induces robust NK 55 cell activation and apoptosis due to triggering of innate nucleic acid sensors, limiting its 56 practicality for mechanistic studies of NK cell biology 8,18,23-28 . Further, in clinical applications, 57 viral transduction carries the risks of sustained adverse effects and oncogenic potential ...

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Polymer-stabilized Cas9 nanoparticles and modified repair templates increase genome editing efficiency

Cited by 213 publications

References 17 publications

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

Efficient Generation of Isogenic Primary Human Myeloid Cells using CRISPR-Cas9 Ribonucleoproteins

TCF-1 regulates the stem-like memory potential of HIV-specific CD8+ T cells in elite controllers

Charge-Altering Releasable Transporters Enable Specific Phenotypic Manipulation of Resting Primary Natural Killer Cells

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