scholarly journals ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

PLoS Biology ◽  
2020 ◽  
Vol 18 (4) ◽  
pp. e3000665 ◽  
Author(s):  
Jelmer Willems ◽  
Arthur P. H. de Jong ◽  
Nicky Scheefhals ◽  
Eline Mertens ◽  
Lisa A. E. Catsburg ◽  
...  
Keyword(s):  
Genome Editing ◽  
Epitope Tagging ◽  
Endogenous Proteins

scholarly journals Systematic gene tagging using CRISPR/Cas9 in human stem cells to illuminate cell organization

2017 ◽  
Author(s):  
Brock Roberts ◽  
Amanda Haupt ◽  
Andrew Tucker ◽  
Tanya Grancharova ◽  
Joy Arakaki ◽  
...  
Keyword(s):  
Stem Cells ◽  
Quality Control ◽  
Genome Editing ◽  
Cell Biology ◽  
Cellular Structures ◽  
Human Stem Cells ◽  
Alpha Tubulin ◽  
Junction Protein ◽  
Endogenous Proteins ◽  
Clonal Lines

AbstractWe present a CRISPR/Cas9 genome editing strategy to systematically tag endogenous proteins with fluorescent tags in human inducible pluripotent stem cells. To date we have generated multiple human iPSC lines with GFP tags for 10 proteins representing key cellular structures. The tagged proteins include alpha tubulin, beta actin, desmoplakin, fibrillarin, lamin B1, non-muscle myosin heavy chain IIB, paxillin, Sec61 beta, tight junction protein ZO1, and Tom20. Our genome editing methodology using Cas9 ribonuclear protein electroporation and fluorescence-based enrichment of edited cells resulted in <0.1-24% HDR across all experiments. Clones were generated from each edited population and screened for precise editing. ∼25% of the clones contained precise mono-allelic edits at the targeted locus. Furthermore, 92% (36/39) of expanded clonal lines satisfied key quality control criteria including genomic stability, appropriate expression and localization of the tagged protein, and pluripotency. Final clonal lines corresponding to each of the 10 cellular structures are now available to the research community. The data described here, including our editing protocol, genetic screening, quality control assays, and imaging observations, can serve as an initial resource for genome editing in cell biology and stem cell research.

scholarly journals Multiplex labeling and manipulation of endogenous neuronal proteins using sequential CRISPR/Cas9 gene editing

2022 ◽  
Author(s):  
Wouter J Droogers ◽  
Jelmer Willems ◽  
Harold D MacGillavry ◽  
Arthur PH de Jong
Keyword(s):  
Genome Editing ◽  
Single Molecule ◽  
Time Window ◽  
Single Cells ◽  
Synaptic Proteins ◽  
Flp Recombinase ◽  
Neuronal Proteins ◽  
Endogenous Proteins ◽  
Living Neurons ◽  
High Degree

Recent advances in CRISPR/Cas9-mediated knock-in methods enable labeling of individual endogenous proteins with fluorophores, to determine their spatiotemporal expression in intact biological preparations. However, multiplex knock-in methods remain limited, particularly in postmitotic cells, due to a high degree of crosstalk between genome editing events. We present Conditional Activation of Knock-in Expression (CAKE), which delivers efficient, flexible and accurate multiplex genome editing in neurons. CAKE is based on sequential gRNA expression operated by a Cre- or Flp-recombinase to control the time window for genomic integration of each donor sequence, which diminishes crosstalk between genome editing events. Importantly, CAKE is compatible with multiple CRISPR/Cas9 strategies, and we show the utilization of CAKE for co-localization of various endogenous proteins, including synaptic scaffolds, ion channels and neurotransmitter receptor subunits. Knock-in efficacy was highly sensitive to DNA vector amount, while knock-in crosstalk was dependent on the rate of donor DNA integration and timing of Cre activation. We applied CAKE to study the co-distribution of endogenous synaptic proteins using dual-color single-molecule localization microscopy, and we introduced dimerization modules to acutely control synaptic receptor dynamics in living neurons. Taken together, CAKE is a versatile method for multiplex protein labeling, enabling accurate detection, precise localization and acute manipulation of endogenous proteins in single cells.

scholarly journals ORANGE: A CRISPR/Cas9-based genome editing toolbox for epitope tagging of endogenous proteins in neurons

2019 ◽  
Author(s):  
Jelmer Willems ◽  
Arthur P.H. de Jong ◽  
Nicky Scheefhals ◽  
Harold D. MacGillavry
Keyword(s):  
Genome Editing ◽  
Cell Biology ◽  
Protein Complexes ◽  
Neuronal Cell ◽  
Super Resolution ◽  
Protein Distribution ◽  
Neuronal Processes ◽  
Endogenous Proteins ◽  
Fluorescent Tags ◽  
Super Resolution Microscopy

ABSTRACTThe correct subcellular distribution of protein complexes establishes the complex morphology of neurons and is fundamental to their functioning. Thus, determining the dynamic distribution of proteins is essential to understand neuronal processes. Fluorescence imaging, in particular super-resolution microscopy, has become invaluable to investigate subcellular protein distribution. However, these approaches suffer from the limited ability to efficiently and reliably label endogenous proteins. We developed ORANGE: an Open Resource for the Application of Neuronal Genome Editing, that mediates targeted genomic integration of fluorescent tags in neurons. This toolbox includes a knock-in library for in-depth investigation of endogenous protein distribution, and a detailed protocol explaining how knock-in can be developed for novel targets. In combination with super-resolution microscopy, ORANGE revealed the dynamic nanoscale organization of endogenous neuronal signaling molecules, synaptic scaffolding proteins, and neurotransmitter receptors. Thus, ORANGE enables quantitation of expression and distribution for virtually any protein in neurons at high resolution and will significantly further our understanding of neuronal cell biology.

scholarly journals An Optimized CRISPR/Cas9 Approach for Precise Genome Editing in Neurons

2020 ◽  
Author(s):  
Huaqiang Fang ◽  
Alexei M. Bygrave ◽  
Richard H. Roth ◽  
Richard C. Johnson ◽  
Richard L. Huganir
Keyword(s):  
Genome Editing ◽  
Model Organism ◽  
Synaptic Proteins ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Coding Regions ◽  
Dividing Cells ◽  
Endogenous Proteins ◽  
Super Ecliptic Phluorin

AbstractThe efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two-guides (TKIT) as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labelling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

scholarly journals CRISPR/Cas9-mediated gene knockin in the hydroid Hydractinia symbiolongicarpus

2018 ◽  
Author(s):  
Steven M. Sanders ◽  
Zhiwei Ma ◽  
Julia M. Hughes ◽  
Brooke M. Riscoe ◽  
Gregory A. Gibson ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Elongation Factor ◽  
Exogenous Dna ◽  
Hydractinia Symbiolongicarpus ◽  
Eukaryotic Elongation Factor ◽  
Two Generations ◽  
Genomics Tools ◽  
Endogenous Proteins

AbstractBackgroundHydractinia symbiolongicarpus, a colonial cnidarian, is a tractable model system for many cnidarian-specific and general biological questions. Until recently, tests of gene function in Hydractinia have relied on laborious forward genetic approaches, randomly integrated transgenes, or transient knockdown of mRNAs.ResultsHere, we report the use of CRISPR/Cas9 genome editing to generate targeted genomic insertions in H. symbiolonigcarpus. We used CRISPR/Cas9 to promote homologous recombination of two fluorescent reporters, eGFP and tdTomato, into the Eukaryotic elongation factor 1 alpha (Eef1a) locus. We demonstrate that the transgenes are expressed ubiquitously and are stable over two generations of breeding. We further demonstrate that CRISPR/Cas9 genome editing can be used to mark endogenous proteins with FLAG or StrepII-FLAG affinity tags to enable in vivo and ex vivo protein studies.ConclusionsThis is the first account of CRISPR/Cas9 mediated knockins in Hydractinia and the first example of the germline transmission of a CRISPR/Cas9 inserted transgene in a cnidarian. The ability to precisely insert exogenous DNA into the Hydractinia genome will enable sophisticated genetic studies and further development of functional genomics tools in this understudied cnidarian model.

scholarly journals An optimized CRISPR/Cas9 approach for precise genome editing in neurons

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Huaqiang Fang ◽  
Alexei M Bygrave ◽  
Richard H Roth ◽  
Richard C Johnson ◽  
Richard L Huganir
Keyword(s):  
Genome Editing ◽  
Model Organism ◽  
Synaptic Proteins ◽  
Two Photon Microscopy ◽  
Two Photon ◽  
Coding Regions ◽  
Dividing Cells ◽  
Endogenous Proteins ◽  
Super Ecliptic Phluorin

The efficient knock-in of large DNA fragments to label endogenous proteins remains especially challenging in non-dividing cells such as neurons. We developed Targeted Knock-In with Two (TKIT) guides as a novel CRISPR/Cas9 based approach for efficient, and precise, genomic knock-in. Through targeting non-coding regions TKIT is resistant to INDEL mutations. We demonstrate TKIT labeling of endogenous synaptic proteins with various tags, with efficiencies up to 42% in mouse primary cultured neurons. Utilizing in utero electroporation or viral injections in mice TKIT can label AMPAR subunits with Super Ecliptic pHluorin, enabling visualization of endogenous AMPARs in vivo using two-photon microscopy. We further use TKIT to assess the mobility of endogenous AMPARs using fluorescence recovery after photobleaching. Finally, we show that TKIT can be used to tag AMPARs in rat neurons, demonstrating precise genome editing in another model organism and highlighting the broad potential of TKIT as a method to visualize endogenous proteins.

scholarly journals Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

2008 ◽  
Vol 5 (2) ◽  
pp. 163-165 ◽  
Author(s):  
Xiaodong Zhang ◽  
Chunguang Guo ◽  
Yueting Chen ◽  
Hennady P Shulha ◽  
Michael P Schnetz ◽  
...  
Keyword(s):  
Epitope Tagging ◽  
Genome Wide ◽  
Endogenous Proteins ◽  
Chip Chip
Sign in / Sign up

Export Citation Format

Share Document