Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters

Umkehrer, Christian; Holstein, Felix; Formenti, Laura; Jude, Julian; Froussios, Kimon; Neumann, Tobias; Cronin, Shona M.; Haas, Linda; Lipp, Jesse J.; Burkard, Thomas R; Fellner, Michaela; Wiesner, Thomas; Zuber, Johannes; Obenauf, Anna C.

doi:10.1038/s41587-020-0614-0

Cited by 78 publications

(58 citation statements)

References 48 publications

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“…Evolving CRISPR/Cas9 technology will also make it possible to genetically manipulate somatic cells in juvenile and adult inbred mice. Although tracking tumor heterogeneity and rare cell subpopulations is challenging, the novel methodology CaTCH (CRISPRa tracing of clones in heterogeneous cell populations) provides new opportunities to study clonal evolution in cancers (Umkehrer et al, 2020).…”

Section: Melanoma Challengesmentioning

confidence: 99%

Melanoma models for the next generation of therapies

et al. 2021

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There is a lack of appropriate melanoma models that can be used to evaluate the efficacy of novel therapeutic modalities. Here, we discuss the current state of the art of melanoma models including genetically engineered mouse, patient-derived xenograft, zebrafish, and ex vivo and in vitro models. We also identify five major challenges that can be addressed using such models, including metastasis and tumor dormancy, drug resistance, the melanoma immune response, and the impact of aging and environmental exposures on melanoma progression and drug resistance. Additionally, we discuss the opportunity for building models for rare subtypes of melanomas, which represent an unmet critical need. Finally, we identify key recommendations for melanoma models that may improve accuracy of preclinical testing and predict efficacy in clinical trials, to help usher in the next generation of melanoma therapies.

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Section: Melanoma Challengesmentioning

confidence: 99%

Melanoma models for the next generation of therapies

et al. 2021

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“…This approach relies on the generation of insertion or deletion (indel) mutations by spCas9 nuclease in a target region to shift the translation frame of a reporter cassette, similar to vectors used to monitor gene-editing outcomes [8,19]. An alternative approach would be to use CRISPRa (dCas9-transcriptional activator) to activate marker expression in a barcode-dependent fashion [20][21][22][23]24]. However, we found that a lentiviral transcriptional activation-based reporter lacked specificity, in part due to a high background level of transcription in a fraction of cells subsequent to genomic integration of the reporter (Additional file 1: Fig.…”

Section: Design Of a Retrieval Vector Activated By Frameshift Mutationsmentioning

confidence: 99%

CloneSifter: enrichment of rare clones from heterogeneous cell populations

et al. 2020

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Background Many biological processes, such as cancer metastasis, organismal development, and acquisition of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. While large-scale barcoding with NGS readout has facilitated cellular fitness assessment at the population level, this approach does not support characterization of clones prior to selection. Single-cell genomics methods provide high biological resolution, but are challenging to scale across large populations to probe rare clones and are destructive, limiting further functional analysis of important clones. Results Here, we develop CloneSifter, a methodology for tracking and enriching rare clones throughout their response to selection. CloneSifter utilizes a CRISPR sgRNA-barcode library that facilitates the isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrate that CloneSifter can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1883 in a heterogeneous cell population. Conclusions CloneSifter provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.

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“…Simply, treatment naïve melanoma cells were transduced with the transcriptional activator dCas9‐VPR and unique barcodes fused to an inducible fluorescent reporter tag. Following drug selection, enriched “drug resistant” clones were determined by genomic sequencing and barcode‐complementary sgRNAs were transduced back into a heterogeneous population of melanoma cells [132]. The fluorescent reporter is activated in the clone of interest, which can be isolated by fluorescence‐activated cell sorting and functionally characterized and compared to treatment naïve founder cells.…”

Section: Identifying Novel Tumor Suppressive and Oncogenic Genes Usinmentioning

confidence: 99%

“…Even more sophisticated platforms have been developed recently, employing CRISPRi and CRISPRa, in addition to CRISPRko, and enabling combinatorial libraries (two sgRNAs per cell) to be assessed [130,131]. CRISPRa Tracing of Clones in Heterogeneous cell populations, or CaTCH, is another new platform that builds on lineage tracing that permits functional characterisation and comparison of founder clones with their post-selection counterparts [132]. CaTCH was utilized to discern whether melanoma cells had pre-existing resistance mechanisms to RAF and MEK inhibitors, or whether they acquired resistance during treatment in vivo [132].…”

Section: Insights Into Future Directions Of Crispr Approaches In Immumentioning

confidence: 99%

“…CRISPRa Tracing of Clones in Heterogeneous cell populations, or CaTCH, is another new platform that builds on lineage tracing that permits functional characterisation and comparison of founder clones with their post‐selection counterparts [132]. CaTCH was utilized to discern whether melanoma cells had pre‐existing resistance mechanisms to RAF and MEK inhibitors, or whether they acquired resistance during treatment in vivo [132]. Simply, treatment naïve melanoma cells were transduced with the transcriptional activator dCas9‐VPR and unique barcodes fused to an inducible fluorescent reporter tag.…”

Section: Identifying Novel Tumor Suppressive and Oncogenic Genes Usinmentioning

confidence: 99%

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Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy

et al. 2020

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The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.

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Isolating live cell clones from barcoded populations using CRISPRa-inducible reporters

Cited by 78 publications

References 48 publications

Melanoma models for the next generation of therapies

Melanoma models for the next generation of therapies

CloneSifter: enrichment of rare clones from heterogeneous cell populations

Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy

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