Efficient delivery of nuclease proteins for genome editing in human stem cells and primary cells.


: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.


Nat Protoc


Nature protocols


Liu J,Gaj T,Yang Y,Wang N,Shui S,Kim S,Kanchiswamy CN,Kim JS,Barbas CF 3rd




Has Abstract


2015-11-01 00:00:00














  • Detection of protein SUMOylation in vivo.

    abstract::The small ubiquitin-like modifiers (SUMOs) are posttranslationally conjugated to eukaryotic cellular proteins with generally unpredictable consequences. SUMO substrates are found in many cellular systems, and functional analysis has revealed that substrate SUMOylation often has an important role in their regulation. H...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Tatham MH,Rodriguez MS,Xirodimas DP,Hay RT

    更新日期:2009-01-01 00:00:00

  • Ambient molecular imaging by desorption electrospray ionization mass spectrometry.

    abstract::Desorption electrospray ionization (DESI) allows the direct analysis of ordinary objects or pre-processed samples under ambient conditions. Among other applications, DESI is used to identify and record spatial distributions of lipids and drug molecules in biological tissue sections. This technique does not require sam...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Wiseman JM,Ifa DR,Venter A,Cooks RG

    更新日期:2008-01-01 00:00:00

  • Genome-wide analysis of replication timing by next-generation sequencing with E/L Repli-seq.

    abstract::This protocol is an extension to: Nat. Protoc. 6, 870-895 (2014); doi:10.1038/nprot.2011.328; published online 02 June 2011Cycling cells duplicate their DNA content during S phase, following a defined program called replication timing (RT). Early- and late-replicating regions differ in terms of mutation rates, transcr...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Marchal C,Sasaki T,Vera D,Wilson K,Sima J,Rivera-Mulia JC,Trevilla-García C,Nogues C,Nafie E,Gilbert DM

    更新日期:2018-05-01 00:00:00

  • Chemical synthesis of proteins using peptide hydrazides as thioester surrogates.

    abstract::This protocol provides a detailed procedure for the chemical synthesis of proteins through native chemical ligation of peptide hydrazides. The two crucial stages of this protocol are (i) the solid-phase synthesis of peptide hydrazides via Fmoc chemistry and (ii) the native chemical ligation of peptide hydrazides throu...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Zheng JS,Tang S,Qi YK,Wang ZP,Liu L

    更新日期:2013-12-01 00:00:00

  • Using the rat forced swim test to assess antidepressant-like activity in rodents.

    abstract::The forced swim test (FST) is one of the most commonly used animal models for assessing antidepressant-like behavior. This protocol details using the FST in rats, which takes place over 48 h and is followed by the video analysis of the behavior. The swim test involves the scoring of active (swimming and climbing) or p...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Slattery DA,Cryan JF

    更新日期:2012-05-03 00:00:00

  • Synthesis and site-specific incorporation of a simple fluorescent pyrimidine.

    abstract::We describe procedures for the synthesis of a fluorescent pyrimidine analog and its site-specific incorporation into a DNA oligomer. The 5'-protected and 3'-activated nucleoside 4 is synthesized in three steps with an overall yield of 40%. Site-specific incorporation into a DNA oligomer occurs with greater than 88% co...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Greco NJ,Tor Y

    更新日期:2007-01-01 00:00:00

  • Generation of a transgenic ORFeome library in Drosophila.

    abstract::Overexpression screens can be used to explore gene function in Drosophila melanogaster, but to demonstrate their full potential, comprehensive and systematic collections of fly strains are required. Here we provide a protocol for high-throughput cloning of Drosophila open-reading frames (ORFs) that are regulated by up...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Bischof J,Sheils EM,Björklund M,Basler K

    更新日期:2014-07-01 00:00:00

  • Tools for investigating peptide-protein interactions: peptide incorporation of environment-sensitive fluorophores via on-resin derivatization.

    abstract::This protocol presents the peptide incorporation of environment-sensitive fluorophores derived from the dimethylaminophthalimide family. The procedure utilizes anhydride precursors of 4-dimethylaminophthalimide (4-DMAP) or 6-dimethylaminonaphthalimide (6-DMN), whose syntheses are described in a related protocol from t...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Sainlos M,Imperiali B

    更新日期:2007-01-01 00:00:00

  • High-throughput ballistic injection nanorheology to measure cell mechanics.

    abstract::High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Wu PH,Hale CM,Chen WC,Lee JS,Tseng Y,Wirtz D

    更新日期:2012-01-05 00:00:00

  • The embryonic mouse hindbrain as a qualitative and quantitative model for studying the molecular and cellular mechanisms of angiogenesis.

    abstract::The mouse embryo hindbrain is a robust and adaptable model for studying sprouting angiogenesis. It permits the spatiotemporal analysis of organ vascularization in normal mice and in mouse strains with genetic mutations that result in late embryonic or perinatal lethality. Unlike postnatal models such as retinal angiog...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Fantin A,Vieira JM,Plein A,Maden CH,Ruhrberg C

    更新日期:2013-02-01 00:00:00

  • Multiparameter screening method for developing optimized red-fluorescent proteins.

    abstract::Genetically encoded fluorescent proteins (FPs) are highly utilized in cell biology research to study proteins of interest or signal processes using biosensors. To perform well in specific applications, these FPs require a multitude of tailored properties. It is for this reason that they need to be optimized by using m...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Bindels DS,Postma M,Haarbosch L,van Weeren L,Gadella TWJ Jr

    更新日期:2020-02-01 00:00:00

  • Isolation and subsequent analysis of murine lamina propria mononuclear cells from colonic tissue.

    abstract::Studies on colonic cells in the lamina propria (LP) of mice are important for understanding the cellular and immune responses in the gut, especially in inflammatory bowel diseases (such as morbus crohn and colitis ulcerosa). This protocol details a method to isolate LP cells and characterize freshly isolated cells by ...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Weigmann B,Tubbe I,Seidel D,Nicolaev A,Becker C,Neurath MF

    更新日期:2007-01-01 00:00:00

  • Analysis of mutational spectra by denaturing capillary electrophoresis.

    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


    authors: Ekstrøm PO,Khrapko K,Li-Sucholeiki XC,Hunter IW,Thilly WG

    更新日期:2008-01-01 00:00:00

  • Design of a functional cyclic HSV1-TK reporter and its application to PET imaging of apoptosis.

    abstract::Positron emission tomography (PET) is a sensitive and noninvasive imaging method that is widely used to explore molecular events in living subjects. PET can precisely and quantitatively evaluate cellular apoptosis, which has a crucial role in various physiological and pathological processes. In this protocol, we descr...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Wang Z,Wang F,Hida N,Kiesewetter DO,Tian J,Niu G,Chen X

    更新日期:2015-05-01 00:00:00

  • A protocol for isolation and culture of mesenchymal stem cells from mouse compact bone.

    abstract::Unlike humans, mouse bone marrow-derived mesenchymal stem cells (MSCs) cannot be easily harvested by adherence to plastic owing to the contamination of cultures by hematopoietic cells. The design of the protocol described here is based on the phenomenon that compact bones abound in MSCs and hematopoietic cells exist i...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Zhu H,Guo ZK,Jiang XX,Li H,Wang XY,Yao HY,Zhang Y,Mao N

    更新日期:2010-03-01 00:00:00

  • Multiparametric characterization of rare HIV-infected cells using an RNA-flow FISH technique.

    abstract::Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on c...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Baxter AE,Niessl J,Fromentin R,Richard J,Porichis F,Massanella M,Brassard N,Alsahafi N,Routy JP,Finzi A,Chomont N,Kaufmann DE

    更新日期:2017-10-01 00:00:00

  • Fast and easy phosphopeptide fractionation by combinatorial ERLIC-SCX solid-phase extraction for in-depth phosphoproteome analysis.

    abstract::Mass spectrometry-based phosphoproteomic analysis is a powerful method for gaining a global, unbiased understanding of cellular signaling. Its accuracy and comprehensiveness stands or falls with the quality and choice of the applied phosphopeptide prefractionation strategy. This protocol covers a powerful but simple a...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Zarei M,Sprenger A,Rackiewicz M,Dengjel J

    更新日期:2016-01-01 00:00:00

  • A strategy for co-translational folding studies of ribosome-bound nascent chain complexes using NMR spectroscopy.

    abstract::During biosynthesis on the ribosome, an elongating nascent polypeptide chain can begin to fold, in a process that is central to all living systems. Detailed structural studies of co-translational protein folding are now beginning to emerge; such studies were previously limited, at least in part, by the inherently dyna...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Cassaignau AM,Launay HM,Karyadi ME,Wang X,Waudby CA,Deckert A,Robertson AL,Christodoulou J,Cabrita LD

    更新日期:2016-08-01 00:00:00

  • MicroRNA detection by northern blotting using locked nucleic acid probes.

    abstract::MicroRNAs (miRNAs) are short, about 21 nucleotides in length, noncoding, regulatory RNA molecules representing a new layer in post-transcriptional regulation of gene expression. Intensive miRNA research has necessitated the development of effective miRNA detection methods such as northern analyses, quantitative real-t...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Várallyay E,Burgyán J,Havelda Z

    更新日期:2008-01-01 00:00:00

  • Hydrogenation using iron oxide-based nanocatalysts for the synthesis of amines.

    abstract::In this protocol, we describe the preparation of nanoscale iron oxide-based materials and their use in the catalysis of different hydrogenation reactions. Pyrolysis of a Fe(OAc)2-phenanthroline complex on carbon at 800 °C under argon atmosphere results in the formation of nanoscale Fe2O3 particles surrounded by nitrog...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Jagadeesh RV,Stemmler T,Surkus AE,Junge H,Junge K,Beller M

    更新日期:2015-04-01 00:00:00

  • Spatially resolved proteomic mapping in living cells with the engineered peroxidase APEX2.

    abstract::This protocol describes a method to obtain spatially resolved proteomic maps of specific compartments within living mammalian cells. An engineered peroxidase, APEX2, is genetically targeted to a cellular region of interest. Upon the addition of hydrogen peroxide for 1 min to cells preloaded with a biotin-phenol substr...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Hung V,Udeshi ND,Lam SS,Loh KH,Cox KJ,Pedram K,Carr SA,Ting AY

    更新日期:2016-03-01 00:00:00

  • Template-based protein structure modeling using the RaptorX web server.

    abstract::A key challenge of modern biology is to uncover the functional role of the protein entities that compose cellular proteomes. To this end, the availability of reliable three-dimensional atomic models of proteins is often crucial. This protocol presents a community-wide web-based method using RaptorX (http://raptorx.uch...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Källberg M,Wang H,Wang S,Peng J,Wang Z,Lu H,Xu J

    更新日期:2012-07-19 00:00:00

  • Generation of cortical neurons from mouse embryonic stem cells.

    abstract::Embryonic stem cells (ESCs) constitute a tool of great potential in neurobiology, enabling the directed differentiation of specific neural cell types. We have shown recently that neurons of the cerebral cortex can be generated from mouse ESCs cultured in a chemically defined medium that contains no morphogen, but in t...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Gaspard N,Bouschet T,Herpoel A,Naeije G,van den Ameele J,Vanderhaeghen P

    更新日期:2009-01-01 00:00:00

  • Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis.

    abstract::We report here a high-throughput method for the modification of bacterial artificial chromosomes (BACs) that uses a novel two-plasmid approach. In this protocol, a vector modified in our laboratory to hold an R6Kγ origin of replication and a marker recombination cassette is inserted into a BAC in a single recombinatio...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Gong S,Kus L,Heintz N

    更新日期:2010-09-01 00:00:00

  • Genetically modified mouse models to help fight COVID-19.

    abstract::The research community is in a race to understand the molecular mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, to repurpose currently available antiviral drugs and to develop new therapies and vaccines against coronavirus disease 2019 (COVID-19). One major challenge in achieving ...

    journal_title:Nature protocols

    pub_type: 杂志文章,评审


    authors: Gurumurthy CB,Quadros RM,Richardson GP,Poluektova LY,Mansour SL,Ohtsuka M

    更新日期:2020-12-01 00:00:00

  • A protocol for phenotypic detection and characterization of vascular cells of different origins in a lung neovascularization model in rodents.

    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


    authors: Jones RC,Capen DE,Cohen KS,Munn LL,Jain RK,Duda DG

    更新日期:2008-01-01 00:00:00

  • Count-based differential expression analysis of RNA sequencing data using R and Bioconductor.

    abstract::RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations) while opt...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Anders S,McCarthy DJ,Chen Y,Okoniewski M,Smyth GK,Huber W,Robinson MD

    更新日期:2013-09-01 00:00:00

  • Generating high-purity cardiac and endothelial derivatives from patterned mesoderm using human pluripotent stem cells.

    abstract::Human pluripotent stem cells (hPSCs) provide a valuable model for the study of human development and a means to generate a scalable source of cells for therapeutic applications. This protocol specifies cell fate efficiently into cardiac and endothelial lineages from hPSCs. The protocol takes 2 weeks to complete and re...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Palpant NJ,Pabon L,Friedman CE,Roberts M,Hadland B,Zaunbrecher RJ,Bernstein I,Zheng Y,Murry CE

    更新日期:2017-01-01 00:00:00

  • In vivo single-cell electroporation for transfer of DNA and macromolecules.

    abstract::Single-cell electroporation allows transfection of plasmid DNA or macromolecules into individual living cells using modified patch electrodes and common electrophysiological equipment. This protocol is optimized for rapid in vivo electroporation of Xenopus laevis tadpole brains with DNA, dextrans, morpholinos and comb...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Bestman JE,Ewald RC,Chiu SL,Cline HT

    更新日期:2006-01-01 00:00:00

  • Intravascular staining for discrimination of vascular and tissue leukocytes.

    abstract::Characterization of the cellular participants in tissue immune responses is crucial to understanding infection, cancer, autoimmunity, allergy, graft rejection and other immunological processes. Previous reports indicate that leukocytes in lung vasculature fail to be completely removed by perfusion. Several studies sug...

    journal_title:Nature protocols

    pub_type: 杂志文章


    authors: Anderson KG,Mayer-Barber K,Sung H,Beura L,James BR,Taylor JJ,Qunaj L,Griffith TS,Vezys V,Barber DL,Masopust D

    更新日期:2014-01-01 00:00:00