CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy

Lambert, M.; Leijonhufvud, Caroline; Segerberg, Filip; Melenhorst, J. Joseph; Carlsten, Mattias

doi:10.1007/978-1-0716-0338-3_18

Cited by 16 publications

(11 citation statements)

References 105 publications

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“…Rapid nucleic acid detection is an important part of many applications in human health and biotechnology, including the identifying of infectious diseases, agricultural pathogens, or circulating DNA or RNA associated with disease. (20-22) CRISPR-Casbased approaches are being tested to treat hereditary, infectious, and many other diseases (23)(24)(25). To date, a number of CRISPR-Cas-based approaches to detect and diagnose infectious and non-infectious diseases (ex: Cancers) had been developed (26)(27)(28).…”

Section: Detection Of Nucleic Acids By Crispr-cas Systemsmentioning

confidence: 99%

SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

2021

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Infectious diseases are one of the most intimidating threats to human race, responsible for an immense burden of disabilities and deaths. Rapid diagnosis and treatment of infectious diseases is a better understanding of its pathogenesis. According to WHO, the ideal approach for detecting foreign pathogens should be rapid, specific, sensitive, instrument-free and cost-effective. Nucleic acid pathogen detection methods, typically PCR have numerous limitations, such as highly sophisticated equipments, reagents, and trained personnel rely on well-established laboratories beside time-consuming. Thus, there is a crucial need to develop novel nucleic acid detection tools with rapid, specific, sensitive, and cost-effective, particularly ones that can be used for versatile point-of-care diagnostic applications. Two new methods exploit on unpredicted in vitro properties CRISPR-Cas effectors, turning activated nucleases into basic amplifiers of a specific nucleic-acid binding event. These effectors are attached with a diversity of reporters and utilized in tandem with present of isothermal amplification approaches to create sensitive identification in multiple field deployable formats. Although still in their beginning, yet SHERLOCK and DETECTR technologies are potential methods for rapid detection and identification of infectious disease, with ultra-sensitive tests that don't require a lot of complicated processing. This review described SHERLOCK and DETECTR technologies beside their properties, functions, and perspectives to become the ultimate diagnostic tools for diagnosing infectious diseases and curbing disease outbreaks.

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Section: Detection Of Nucleic Acids By Crispr-cas Systemsmentioning

confidence: 99%

SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

2021

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“…Recent advances in CRISPR technologies have reinvigorated interest in genome editing of human NK cells ( Rautela et al, 2018 Preprint ; Lambert et al, 2020 ; Kararoudi et al, 2019 Preprint ; Pomeroy et al, 2020 ; Nguyen et al, 2020 ). Cas9 ribonucleoprotein (RNP) is already a popular format for various cell types owing to its transient activity, high efficiency, and multiplex capability ( Farboud et al, 2018 ).…”

Section: Introductionmentioning

confidence: 99%

A robust platform for expansion and genome editing of primary human natural killer cells

Huang

Lai

Shih

et al. 2021

Journal of Experimental Medicine

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Genome editing is a powerful technique for delineating complex signaling circuitry and enhancing the functionality of immune cells for immunotherapy. Natural killer (NK) cells are potent immune effectors against cell malignancy, but they are challenging to modify genetically by conventional methods due to the toxicity of DNA when introduced into cells coupled with limited transfection and transduction efficiency. Here, we describe an integrated platform that streamlines feeder-free ex vivo expansion of cryopreserved primary human NK cells and nonviral genome editing by the nucleofection of CRISPR-Cas9 ribonucleoproteins (Cas9 RNPs). The optimized Cas9 nucleofection protocol allows efficient and multiplex gene knockout in NK cells while preserving high cell viability and negligible off-target effects. Cointroduction of a DNA template also enables in-frame gene knock-in of an HA affinity tag and a gfp reporter across multiple loci. This work demonstrates the advantages and flexibility of working with cryopreserved NK cells as potential off-the-shelf engineered therapeutic agents.

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“…However, several of these cell therapies remain unpredictable in terms of efficacy and potential side effects. A change of strategies is required in order to improve efficacy while minimising the risk of complications [ 11 – 13 ]. With the rapid increase of new and more targeted cell therapies, it is essential to develop reliable tools to better understand the biodistribution of the cell therapy product once infused in vivo [ 12 , 13 ].…”

Section: Introductionmentioning

confidence: 99%

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

et al. 2021

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Background There is a need to better characterise cell-based therapies in preclinical models to help facilitate their translation to humans. Long-term high-resolution tracking of the cells in vivo is often impossible due to unreliable methods. Radiolabelling of cells has the advantage of being able to reveal cellular kinetics in vivo over time. This study aimed to optimise the synthesis of the radiotracers [89Zr]Zr-oxine (8-hydroxyquinoline) and [89Zr]Zr-DFO-NCS (p-SCN-Bn-Deferoxamine) and to perform a direct comparison of the cell labelling efficiency using these radiotracers. Procedures Several parameters, such as buffers, pH, labelling time and temperature, were investigated to optimise the synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS in order to reach a radiochemical conversion (RCC) of >95 % without purification. Radio-instant thin-layer chromatography (iTLC) and radio high-performance liquid chromatography (radio-HPLC) were used to determine the RCC. Cells were labelled with [89Zr]Zr-oxine or [89Zr]Zr-DFO-NCS. The cellular retention of 89Zr and the labelling impact was determined by analysing the cellular functions, such as viability, proliferation, phagocytotic ability and phenotypic immunostaining. Results The optimised synthesis of [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS resulted in straightforward protocols not requiring additional purification. [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS were synthesised with an average RCC of 98.4 % (n = 16) and 98.0 % (n = 13), respectively. Cell labelling efficiencies were 63.9 % (n = 35) and 70.2 % (n = 30), respectively. 89Zr labelling neither significantly affected the cell viability (cell viability loss was in the range of 1–8 % compared to its corresponding non-labelled cells, P value > 0.05) nor the cells’ proliferation rate. The phenotype of human decidual stromal cells (hDSC) and phagocytic function of rat bone-marrow-derived macrophages (rMac) was somewhat affected by radiolabelling. Conclusions Our study demonstrates that [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS are equally effective in cell labelling. However, [89Zr]Zr-oxine was superior to [89Zr]Zr-DFO-NCS with regard to long-term stability, cellular retention, minimal variation between cell types and cell labelling efficiency.

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CRISPR/Cas9-Based Gene Engineering of Human Natural Killer Cells: Protocols for Knockout and Readouts to Evaluate Their Efficacy

Cited by 16 publications

References 105 publications

SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases

A robust platform for expansion and genome editing of primary human natural killer cells

Optimisation of the Synthesis and Cell Labelling Conditions for [89Zr]Zr-oxine and [89Zr]Zr-DFO-NCS: a Direct In Vitro Comparison in Cell Types with Distinct Therapeutic Applications

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