Abstract:
:Targeted nucleases, including zinc-finger nucleases (ZFNs), transcription activator-like (TAL) effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9), have provided researchers with the ability to manipulate nearly any genomic sequence in human cells and model organisms. However, realizing the full potential of these genome-modifying technologies requires their safe and efficient delivery into relevant cell types. Unlike methods that rely on expression from nucleic acids, the direct delivery of nuclease proteins to cells provides rapid action and fast turnover, leading to fewer off-target effects while maintaining high rates of targeted modification. These features make nuclease protein delivery particularly well suited for precision genome engineering. Here we describe procedures for implementing protein-based genome editing in human embryonic stem cells and primary cells. Protocols for the expression, purification and delivery of ZFN proteins, which are intrinsically cell-permeable; TALEN proteins, which can be internalized via conjugation with cell-penetrating peptide moieties; and Cas9 ribonucleoprotein, whose nucleofection into cells facilitates rapid induction of multiplexed modifications, are described, along with procedures for evaluating nuclease protein activity. Once they are constructed, nuclease proteins can be expressed and purified within 6 d, and they can be used to induce genomic modifications in human cells within 2 d.
journal_name
Nat Protocjournal_title
Nature protocolsauthors
Liu J,Gaj T,Yang Y,Wang N,Shui S,Kim S,Kanchiswamy CN,Kim JS,Barbas CF 3rddoi
10.1038/nprot.2015.117subject
Has Abstractpub_date
2015-11-01 00:00:00pages
1842-59issue
11eissn
1754-2189issn
1750-2799pii
nprot.2015.117journal_volume
10pub_type
杂志文章相关文献
Nature Protocols文献大全abstract::Here we present a protocol for the extraction of RNA from Dictyostelium discoideum. Dictyostelium is a social amoeba that undergoes a basic developmental program, and therefore analysis of RNA levels over a time course is a commonly used technique. This procedure is similar to other guanidine thiocyanate-based methods...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.191
更新日期:2007-01-01 00:00:00
abstract::Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.92
更新日期:2006-01-01 00:00:00
abstract::This protocol describes a simple but robust microfluidic assay combining three-dimensional (3D) and two-dimensional (2D) cell culture. The microfluidic platform comprises hydrogel-incorporating chambers between surface-accessible microchannels. By using this platform, well-defined biochemical and biophysical stimuli c...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2012.051
更新日期:2012-06-07 00:00:00
abstract::This protocol describes a methodology for imaging the sequestration of infected erythrocytes of the rodent malaria parasite Plasmodium berghei in the bodies of live mice or in dissected organs, using a transgenic parasite that expresses luciferase. Real-time imaging of infected erythrocytes is performed by measuring b...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.69
更新日期:2006-01-01 00:00:00
abstract::This protocol describes a method for the extraction of DNA from elephant ivory. These techniques are being used to assign geographic origin to poached ivory by comparing the ivory genotype to a geographic-based gene frequency map, developed separately. The method has three components: ivory pulverization, decalcificat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.318
更新日期:2007-01-01 00:00:00
abstract::The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.239
更新日期:2006-01-01 00:00:00
abstract::This protocol describes an efficient method to site-specifically label cell-surface or purified proteins with chemical probes in two steps: probe incorporation mediated by enzymes (PRIME) followed by chelation-assisted copper-catalyzed azide-alkyne cycloaddition (CuAAC). In the PRIME step, Escherichia coli lipoic acid...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.096
更新日期:2013-08-01 00:00:00
abstract::To uncover the function of and interplay between the mammalian cytosine modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), new techniques and advances in current technology are needed. To this end, we have developed oxidative bisulfite sequencing (oxBS-seq), which can quantitatively locate 5mC an...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.115
更新日期:2013-10-01 00:00:00
abstract::The Buccal Micronucleus Cytome (BMCyt) assay is a minimally invasive method for studying DNA damage, chromosomal instability, cell death and the regenerative potential of human buccal mucosal tissue. This method is increasingly used in molecular epidemiological studies for investigating the impact of nutrition, lifest...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.53
更新日期:2009-01-01 00:00:00
abstract::The goal of many current studies of neovascularization is to define the phenotype of vascular cell populations of different origins and to determine how such cells promote assembly of vascular channel. Here, we describe a protocol to immunophenotype vascular cells by high-resolution imaging and by fluorescence-activat...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2007.537
更新日期:2008-01-01 00:00:00
abstract::Post-translational protein nitration has attracted interest owing to its involvement in cellular signaling, effects on protein function and potential as biomarker of nitroxidative stress. We describe a procedure for enriching nitropeptides for mass spectrometry (MS)-based proteomics that is a simple and reliable alter...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2014.052
更新日期:2014-04-01 00:00:00
abstract::The unprecedented increase in the throughput of DNA sequencing driven by next-generation technologies now allows efficient analysis of the complete protein-coding regions of genomes (exomes) for multiple samples in a single sequencing run. However, sample preparation and targeted enrichment of multiple samples has bec...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.396
更新日期:2011-11-03 00:00:00
abstract::Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and p...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2016.036
更新日期:2016-04-01 00:00:00
abstract::Conventional gene expression studies analyze multiple cells simultaneously or single cells, for which the exact in vivo or in situ position is unknown. Although cellular heterogeneity can be discerned when analyzing single cells, any spatially defined attributes that underpin the heterogeneous nature of the cells cann...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.003
更新日期:2017-03-01 00:00:00
abstract::Small ubiquitin-like modifier (SUMO) post-translational modification (PTM) of proteins has a crucial role in the regulation of important cellular processes. This protocol describes the chemical synthesis of functional SUMO-peptide conjugates. The two crucial stages of this protocol are the solid-phase synthesis of pep...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2015.013
更新日期:2015-02-01 00:00:00
abstract::The point mutational spectrum over nearly any 75- to 250-bp DNA sequence isolated from cells, tissues or large populations may be discovered using denaturing capillary electrophoresis (DCE). A modification of the standard DCE method that uses cycling temperature (e.g., +/-5 degrees C), CyDCE, permits optimal resolutio...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.79
更新日期:2008-01-01 00:00:00
abstract::Photoconvertible fluorescent proteins, such as Kaede, can be switched irreversibly from their native color to a new one. This property can be exploited to visualize de novo mRNA translation, because newly synthesized proteins can be distinguished from preexisting ones by their color. In this protocol, Kaede cDNA linke...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2008.113
更新日期:2008-01-01 00:00:00
abstract::Brachypodium distachyon is emerging as a new model system for bridging research into temperate cereal crops, such as wheat and barley, and for promoting research in novel biomass grasses. Here, we provide an adapter ligation PCR protocol that allows the large-scale characterization of T-DNA insertions into the genome ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2009.32
更新日期:2009-01-01 00:00:00
abstract::This protocol describes the efficient, generally applicable Ullmann coupling reaction of bromaminic acid with alkyl- or aryl-amines in phosphate buffer under microwave irradiation using elemental copper as a catalyst. The reaction leads to a number of biologically active compounds. As a prototypical example, the synth...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2010.63
更新日期:2010-05-01 00:00:00
abstract::Precise identification of sites of RNA modification is key to studying the functional role of such modifications in the regulation of gene expression and for elucidating relevance to diverse physiological processes. tRNA reduction and cleavage sequencing (TRAC-Seq) is a chemically based approach for the unbiased globa...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-019-0226-7
更新日期:2019-11-01 00:00:00
abstract::Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in com...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.263
更新日期:2006-01-01 00:00:00
abstract::This protocol describes an optimized method for direct in vitro monitoring of homo- and heterotypic axon-axon interactions involved in the developmental assembly of neural circuits. The assay exploits a classical example of heterotypic axonal interactions by modeling the sequential extension of spinal motor and somato...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2011.442
更新日期:2012-01-26 00:00:00
abstract::Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2006.17
更新日期:2006-01-01 00:00:00
abstract::The CRISPR-Cas9 genome-editing tool and the availability of whole-genome sequences from plant species have revolutionized our ability to introduce targeted mutations into important crop plants, both to explore genetic changes and to introduce new functionalities. Here, we describe protocols adapting the CRISPR-Cas9 sy...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0067-9
更新日期:2018-12-01 00:00:00
abstract::This protocol measures externalization of aminophospholipids (APLs) to the outside of the plasma membrane using mass spectrometry (MS). APL externalization occurs in numerous events, and it is relevant for transplant medicine, immunity and cancer. In this protocol, externalized APLs are chemically modified by using a ...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.163
更新日期:2014-01-01 00:00:00
abstract::The genomic instability of stem cells in culture, caused by their routine in vitro propagation or by their genetic manipulation, is deleterious both for their clinical application and for their use in basic research. Frequent evaluation of the genomic integrity of stem cells is thus required, and it is usually perform...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.051
更新日期:2013-05-01 00:00:00
abstract::Coimmunoprecipitation (co-IP) analysis is a useful method for studying protein-protein interactions. It currently involves electrophoresis and western blotting, which are not optimized for detecting weak and transient interactions. In this protocol we describe an advanced version of co-IP analysis that uses real-time,...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2013.116
更新日期:2013-10-01 00:00:00
abstract::Growing evidence indicates that RNA G-quadruplexes have important roles in various processes such as transcription, translation, regulation of telomere length, and formation of telomeric heterochromatin. Investigation of RNA G-quadruplex structures associated with biological events is therefore essential to understand...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.156
更新日期:2018-04-01 00:00:00
abstract::Fast, high-resolution mapping of heterogeneous interfaces with a wide elastic modulus range is a major goal of atomic force microscopy (AFM). This goal becomes more challenging when the nanomechanical mapping involves biomolecules in their native environment. Over the years, several AFM-based methods have been develop...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/s41596-018-0070-1
更新日期:2018-12-01 00:00:00
abstract::Protein ADP-ribosylation is a structurally heterogeneous post-translational modification (PTM) that influences the physicochemical and biological properties of the modified protein. ADP-ribosylation of chromatin changes its structural properties, thereby regulating important nuclear functions. A lack of suitable antib...
journal_title:Nature protocols
pub_type: 杂志文章
doi:10.1038/nprot.2017.072
更新日期:2017-09-01 00:00:00