Abstract:CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded … Show more
“…For instance, Cas9 variants with higher specificity could decrease indel frequency [66,67], and the knock-in efficiency and repair accuracy may be predicted based on the target sequence [28,29]. Also, alternative delivery methods such as ribonucleoproteins (RNPs) [68] might increase the efficiency of DNA delivery.…”
The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knockins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.
“…For instance, Cas9 variants with higher specificity could decrease indel frequency [66,67], and the knock-in efficiency and repair accuracy may be predicted based on the target sequence [28,29]. Also, alternative delivery methods such as ribonucleoproteins (RNPs) [68] might increase the efficiency of DNA delivery.…”
The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knockins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.
“…For instance, Cas9 variants that are more accurate could increase specificity and decrease indel frequency (50,51), and the indel frequency may be predicted based on the target sequence (25,26). Also, alternative delivery methods such as ribonucleoproteins (RNPs) (52), might increase the efficiency of targeted integration.…”
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.
“…As E2A is regulated by Id1 on the protein level, rather than the transcriptional level, we generated an endogenously tagged ES cell line ( Figure 1B) using CRISPR/Cas9 targeting to follow the expression of E2A protein during differentiation. Based on a strategy previously developed to tag neural stem cells with high efficiency (Dewari et al, 2018), guide RNA (sgRNA) was designed to cut proximal to the stop codon in the 3'UTR of E2A (Supplementary Figure 1A), and was co-transfected into wild-type ES cells with recombinant Cas9 protein (rCas9) and a single-stranded donor DNA template (ssODN) encoding the V5 tag flanked by homology arms (Supplementary Figure 1B ).…”
Section: E2a Is Expressed Heterogeneously In Pluripotent Es Cells Andmentioning
confidence: 99%
“…For ssODN design, the V5 tag sequence was flanked by ~75bp homology arms and a PAM-blocking mutation (NGG>NGC) was introduced into the 3'UTR sequence. Ribonucleoprotein (RNP) complexes were assembled as described in (Dewari et al, 2018). Briefly, crRNA and tracrRNA oligos were mixed in equimolar concentration, heated at 95°C for 5 min and allowed to cool to room temperature to anneal.…”
Section: Crispr/cas9 Epitope Tagging Of Es Cell Linesmentioning
The intrinsic mechanisms that link extracellular signalling to the onset of neural differentiation are not well understood. In pluripotent mouse cells, BMP blocks entry into the neural lineage via transcriptional upregulation of Inhibitor of Differentiation (Id) factors. We have previously identified that the major binding partner of Id proteins in pluripotent cells is the basic helix-loop-helix (bHLH) transcription factor (TF), E2A. Id1 can prevent E2A from forming heterodimers with bHLH TFs or from forming homodimers. Here, we show that overexpression of a forced E2A homodimer is sufficient to drive robust neural commitment in pluripotent cells, even under non-permissive conditions. Conversely, we find that E2A null cells display a defect in their neural differentiation capacity. E2A acts as an upstream activator of neural lineage genes, including Sox1 and Foxd4, and as a repressor of Nodal signalling. Our results suggest a crucial role for E2A in establishing neural lineage commitment in pluripotent cells.
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