Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Wroblewska, Aleksandra; Dhainaut, Maxime; Ben-Zvi, Benjamin; Rose, Samuel A.; Park, Eun Sook; Amir, El-ad David; Bektesevic, Anela; Baccarini, Alessia; Mérad, Miriam; Rahman, Adeeb; Brown, Brian D.

doi:10.1016/j.cell.2018.09.022

Cited by 126 publications

(110 citation statements)

References 41 publications

(63 reference statements)

Supporting

Mentioning

100

Contrasting

Order By: Relevance

“…This was recently highlighted in a novel approach to CRISPR genomics where expression of sgRNAs was coupled with specific protein barcodes, allowing for simultaneous multidimensional phenotypic analysis of several dozen knockouts at a single-cell resolution [48]. In a pooled parallel analysis of gene editing efficiency for ten genes (3–4 sgRNAs per gene), the authors demonstrated that the gene knockout at the protein level was highly variable depending on the sgRNAs used.…”

Section: Guide Rna Efficiency and Specificitymentioning

confidence: 99%

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

et al. 2019

Self Cite

View full text Add to dashboard Cite

CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

show abstract

Section: Guide Rna Efficiency and Specificitymentioning

confidence: 99%

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

et al. 2019

Self Cite

View full text Add to dashboard Cite

show abstract

“…The performance of sgRNAs targeting the same gene can vary dramatically. This was recently highlighted in a novel approach to CRISPR genomics where expression of sgRNAs was coupled with specific protein barcodes, allowing for simultaneous multidimensional phenotypic analysis of several dozens of knockouts at a single cell resolution (Wroblewska et al, 2018). In a pooled parallel analysis of gene editing efficiency for 10 genes (3-4 sgRNAs guide per gene), the authors demonstrated that the gene KO at the protein level was highly variable depending on the sgRNAs used.…”

Section: Guide Rna Efficiency and Specificitymentioning

confidence: 99%

Towards best-practice approaches for CRISPR/Cas9 gene engineering

Campenhout

Cabochette

Veillard

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

In recent years, CRISPR has evolved from "the curious sequence of unknown biological function" into a functional genome editing tool. The CRISPR/Cas9 technology is now delivering novel genetic models for fundamental research, drug screening, therapy development, rapid diagnostics and transcriptional modulation. Despite the apparent simplicity of the CRISPR/Cas9 system, the outcome of a genome editing experiment can be substantially impacted by technical parameters as well as biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome targeting efficiency and specificity.The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome editing experiments.2

show abstract

“…Simultaneous with this paper, a method termed Pro-Code that utilises plasma membrane-based epitope barcodes was published. 12 Pro-Code enables highly multiplexed protein-based barcoding of cell clones and was applied in conjunction with single-cell CRISPR screens using flow-based mass cytometry. Although suitable for analysis of clonal dynamics and signalling states at the single cell level in cellular suspensions, such methods are not useful for spatial compartmentalisation and imaging of barcoded clones within complex cellular mixtures or tissues due to close cell-cell membrane contacts resulting in overlapping, indistinguishable cell populations.…”

Section: Introductionmentioning

confidence: 99%

Resolving Tumour Clonal Heterogeneity and Spatial Complexity using Nuclear Tandem Epitope Protein (nTEP) Barcoding

Gill

Koplev

Machel

et al. 2018

Preprint

View full text Add to dashboard Cite

Tumours are composed of an array of unique cancer cell clones along with many non-tumour cells such as immune cells, fibroblasts and endothelial cells, which make up the complex tumour microenvironment. To better understand the co-evolution of tumour clones and cells of the tumour microenvironment, we require tools to spatially resolve heterotypic cellular interactions at the single cell level. We present a novel protein-based barcoding technology termed nuclear tandem epitope protein (nTEP) barcoding, which can be designed to combinatorially encode and track dozens to hundreds of tumour clones in their spatial context within complex cellular mixtures using multiplexed antibody-based imaging. Here we provide proof-of-principle of nTEP barcoding and develop the technology, which relies on lentiviralbased stable expression of a nuclear-localised fluorophore that contains unique combinations of protein epitope tags that can be decoded by a limited set of antibodies. By generating a series of cell lines expressing unique nTEP barcodes, we were able to robustly identify and spatially deconvolve specific clones present within highly complex cell mixtures at the single cell level using state-of-the-art iterative indirect immunofluorescence imaging (4i). We define the utility of nTEP-barcoding as a powerful tool for visualising and resolving tumour heterogeneity at the cellular level, and envision its usage in mouse tumour models for understanding how tumour clones modulate and interact with stromal-and immune cells in cancer.

show abstract

Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens

Cited by 126 publications

References 41 publications

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

Towards best-practice approaches for CRISPR/Cas9 gene engineering

Resolving Tumour Clonal Heterogeneity and Spatial Complexity using Nuclear Tandem Epitope Protein (nTEP) Barcoding

Contact Info

Product

Resources

About