Methods in enzymology
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The rapid advancement of genome-editing techniques holds much promise for the field of human gene therapy. From bacteria to model organisms and human cells, genome editing tools such as zinc-finger nucleases (ZNFs), TALENs, and CRISPR/Cas9 have been successfully used to manipulate the respective genomes with unprecedented precision. With regard to human gene therapy, it is of great interest to test the feasibility of genome editing in primary human hematopoietic cells that could potentially be used to treat a variety of human genetic disorders such as hemoglobinopathies, primary immunodeficiencies, and cancer. ⋯ By using two guide RNAs directed at a single locus, we achieve highly efficient and predictable deletions that ablate gene function. The use of a Cas9-2A-GFP fusion protein allows FACS-based enrichment of the transfected cells. The ease of designing, constructing, and testing guide RNAs makes this dual guide strategy an attractive approach for the efficient deletion of clinically relevant genes in primary human hematopoietic stem and effector cells and enables the use of CRISPR/Cas9 for gene therapy.
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Methods in enzymology · Jan 2014
Use of safranin for the assessment of mitochondrial membrane potential by high-resolution respirometry and fluorometry.
The mitochondrial transmembrane potential (Δψmt or mtMP) is directly influenced by oxidative phosphorylation (OXPHOS). The exact nature of the interactions between respiration (flux) and mtMP (force) under various physiological and pathological conditions remains unclear, partially due to methodological limitations. Here, we describe a combination of high-resolution respirometry and fluorometry based on the OROBOROS Oxygraph-2k and the widely applied mtMP indicator safranin. ⋯ Taken together, the combined measurement of respiration and mtMP greatly enhances the informative potential of OXPHOS studies. The respirometric validation of inhibitory and uncoupling effects is mandatory for any fluorophore employed to assess mtMP in any respiratory state, tissue type, and pathophysiological condition. The methodological issues analyzed herein are relevant for the study of mitochondrial respiration in a wide variety of setting, including cancer cell metabolism.
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Methods in enzymology · Jan 2014
The iCRISPR platform for rapid genome editing in human pluripotent stem cells.
Human pluripotent stem cells (hPSCs) have the potential to generate all adult cell types, including rare or inaccessible human cell populations, thus providing a unique platform for disease studies. To realize this promise, it is essential to develop methods for efficient genetic manipulations in hPSCs. Established using TALEN (transcription activator-like effector nuclease) and CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) systems, the iCRISPR platform supports a variety of genome-engineering approaches with high efficiencies. ⋯ We have successfully used these protocols in four different hPSC lines, including human embryonic stem cells and induced pluripotent stem cells. Once the iCRISPR platform is established, clonal lines with desired genetic modifications can be established in as little as 1 month. The methods described here enable a wide range of genome-engineering applications in hPSCs, thus providing a valuable resource for the creation of diverse hPSC-based disease models with superior speed and ease.
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Methods in enzymology · Jan 2014
Methods to monitor ROS production by fluorescence microscopy and fluorometry.
Mitochondria are considered one of the main sources of reactive oxygen species (ROS). The overgeneration of ROS can evoke an intracellular state of oxidative stress, leading to permanent cell damage. ⋯ Here, we describe the use of chemical probes for the rapid detection of ROS in intact and permeabilized adherent cells by fluorescence microscopy and fluorometry. Moreover, after discussing the limitations described in the literature for the fluorescent probes presented herein, we recommend methods to assess the production of specific ROS in various fields of investigation, including the study of oncometabolism.
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Methods in enzymology · Jan 2014
Targeted genome editing in human cells using CRISPR/Cas nucleases and truncated guide RNAs.
CRISPR RNA-guided nucleases have recently emerged as a robust genome-editing platform that functions in a wide range of organisms. To reduce off-target effects of these nucleases, we developed and validated a modified system that uses truncated guide RNAs (tru-gRNAs). The use of tru-gRNAs leads to decreases in off-target effects and does not generally compromise the on-target efficiencies of these genome-editing nucleases. In this chapter, we describe guidelines for identifying potential tru-gRNA target sites and protocols for measuring the on-target efficiencies of CRISPR RNA-guided nucleases in human cells.