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 Early alphavirus replication dynamics in single cells reveal a passive basis for superinfection exclusion

2020 ◽  
Author(s):  
Zakary S. Singer ◽  
Pradeep M. Ambrose ◽  
Tal Danino ◽  
Charles M. Rice
Keyword(s):  
Single Molecule ◽  
Time Window ◽  
Single Cells ◽  
Viral Kinetics ◽  
Protein Kinetics ◽  
Superinfection Exclusion ◽  
Viral Replicase ◽  
Competitive Phenotypes ◽  
Over Time

SummaryWhile decades of research have elucidated many steps in the alphavirus lifecycle, the earliest replication dynamics have remained unclear. This missing time window has obscured early replicase strand synthesis behavior and prevented elucidation of how the resulting activity gives rise to a superinfection exclusion environment, one of the fastest competitive phenotypes among viruses. Using quantitative live-cell and single-molecule imaging, we characterize the strand preferences of the viral replicase in situ, and measure protein kinetics in single cells over time. In this framework, we evaluate competition between alphaviruses, and uncover that early superinfection exclusion is actually not a binary and unidirectional process, but rather a graded and bidirectional viral interaction. In contrast to competition between other viruses, alphaviruses demonstrate a passive basis for superinfection exclusion, emphasizing the utility of analyzing viral kinetics within single cells.

scholarly journals CD-tagging-MS2: detecting allelic expression of endogenous mRNAs and their protein products in single cells

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Jonathan Sheinberger ◽  
Hodaya Hochberg ◽  
Erez Lavi ◽  
Itamar Kanter ◽  
Shira Avivi ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Cell Clone ◽  
Single Cells ◽  
Simultaneous Detection ◽  
Protein Product ◽  
Intact Cells ◽  
Endogenous Gene ◽  
Specific Allele ◽  
Endogenous Proteins

Abstract Discriminating between the mRNA and protein outputs of each of the alleles of an endogenous gene in intact cells, is a difficult task. To examine endogenous transcripts originating from a specific allele, we applied Central Dogma tagging (CD-tagging), which is based on a tag insertion into an endogenous gene by creation of a new exon. Previously, CD-tagging was used to tag endogenous proteins. Here we developed a CD-tagging-MS2 approach in which two tags were inserted in tandem; a fluorescent protein tag in conjunction with the mRNA MS2 tag used for tagging mRNAs in cells. A cell clone library of CD-tagged-MS2 genes was generated, and protein and mRNA distributions were examined and characterized in single cells. Taking advantage of having one allele tagged, we demonstrate how the transcriptional activity of all alleles, tagged and untagged, can be identified using single molecule RNA fluorescence in situ hybridization (smFISH). Allele-specific mRNA expression and localization were quantified under normal and stress conditions. The latter generate cytoplasmic stress granules (SGs) that can store mRNAs, and the distribution of the mRNAs within and outside of the SGs was measured. Altogether, CD-tagging-MS2 is a robust and inexpensive approach for direct simultaneous detection of an endogenous mRNA and its translated protein product in the same cell.

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 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 Highly specific imaging of mRNA in single cells by target RNA-initiated rolling circle amplification

2017 ◽  
Vol 8 (5) ◽  
pp. 3668-3675 ◽  
Author(s):  
Ruijie Deng ◽  
Kaixiang Zhang ◽  
Yupeng Sun ◽  
Xiaojun Ren ◽  
Jinghong Li
Keyword(s):  
Single Molecule ◽  
Single Cells ◽  
Rolling Circle Amplification ◽  
Robust Method ◽  
Single Nucleotide ◽  
Rolling Circle ◽  
Target Rna

We report a robust method for the efficient imaging of mRNA with single-nucleotide and near-single-molecule resolution in single cells.

scholarly journals RA3 is a reference-guided approach for epigenetic characterization of single cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Shengquan Chen ◽  
Guanao Yan ◽  
Wenyu Zhang ◽  
Jinzhao Li ◽  
Rui Jiang ◽  
...  
Keyword(s):  
Single Cell ◽  
Computational Analysis ◽  
Reference Data ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Biological Variation ◽  
Superior Performance ◽  
High Dimensionality ◽  
High Degree

AbstractThe recent advancements in single-cell technologies, including single-cell chromatin accessibility sequencing (scCAS), have enabled profiling the epigenetic landscapes for thousands of individual cells. However, the characteristics of scCAS data, including high dimensionality, high degree of sparsity and high technical variation, make the computational analysis challenging. Reference-guided approaches, which utilize the information in existing datasets, may facilitate the analysis of scCAS data. Here, we present RA3 (Reference-guided Approach for the Analysis of single-cell chromatin Accessibility data), which utilizes the information in massive existing bulk chromatin accessibility and annotated scCAS data. RA3 simultaneously models (1) the shared biological variation among scCAS data and the reference data, and (2) the unique biological variation in scCAS data that identifies distinct subpopulations. We show that RA3 achieves superior performance when used on several scCAS datasets, and on references constructed using various approaches. Altogether, these analyses demonstrate the wide applicability of RA3 in analyzing scCAS data.

Reduction in gene expression noise by targeted increase in accessibility at gene loci

2021 ◽  
Vol 118 (42) ◽  
pp. e2018640118
Author(s):  
LaTasha C. R. Fraser ◽  
Ryan J. Dikdan ◽  
Supravat Dey ◽  
Abhyudai Singh ◽  
Sanjay Tyagi
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Single Molecule ◽  
Single Cells ◽  
Global Changes ◽  
Messenger Rnas ◽  
Histone Acetyl Transferase ◽  
Gene Expression Noise ◽  
Expression Noise ◽  
Eukaryotic Genes

Many eukaryotic genes are expressed in randomly initiated bursts that are punctuated by periods of quiescence. Here, we show that the intermittent access of the promoters to transcription factors through relatively impervious chromatin contributes to this “noisy” transcription. We tethered a nuclease-deficient Cas9 fused to a histone acetyl transferase at the promoters of two endogenous genes in HeLa cells. An assay for transposase-accessible chromatin using sequencing showed that the activity of the histone acetyl transferase altered the chromatin architecture locally without introducing global changes in the nucleus and rendered the targeted promoters constitutively accessible. We measured the gene expression variability from the gene loci by performing single-molecule fluorescence in situ hybridization against mature messenger RNAs (mRNAs) and by imaging nascent mRNA molecules present at active gene loci in single cells. Because of the increased accessibility of the promoter to transcription factors, the transcription from two genes became less noisy, even when the average levels of expression did not change. In addition to providing evidence for chromatin accessibility as a determinant of the noise in gene expression, our study offers a mechanism for controlling gene expression noise which is otherwise unavoidable.

scholarly journals Direct observation of frequency modulated transcription in single cells using light activation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Daniel R Larson ◽  
Christoph Fritzsch ◽  
Liang Sun ◽  
Xiuhau Meng ◽  
David S Lawrence ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Cell Model ◽  
Single Cells ◽  
Single Gene ◽  
Gene Activation ◽  
Light Activation ◽  
Dynamic Synthesis ◽  
Single Cell Model

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.

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.

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