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On the design of CRISPR-based single-cell molecular screens.

ABSTRACT: Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ?50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.

SUBMITTER: Hill AJ

PROVIDER: S-EPMC5882576 | biostudies-literature | 2018 Apr

REPOSITORIES: biostudies-literature

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On the design of CRISPR-based single-cell molecular screens.

Hill Andrew J AJ McFaline-Figueroa José L JL Starita Lea M LM Gasperini Molly J MJ Matreyek Kenneth A KA Packer Jonathan J Jackson Dana D Shendure Jay J Trapnell Cole C

Nature methods 20180219 4

Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%. ...[more]

PMID: 29457792

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On the design of CRISPR-based single cell molecular screens

Project description:We demonstrate that vector designs for such screens that rely on cis linkage of guides and distally located barcodes suffer from swapping of intended guide-barcode associations at rates approaching 50% due to template switching during lentivirus production, greatly reducing sensitivity.

2018-01-29 | GSE108699 | GEO

On the design of CRISPR-based single cell molecular screens

Project description:On the design of CRISPR-based single cell molecular screens

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Protein Barcodes Enable High-Dimensional Single-Cell CRISPR Screens.

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GiRAFR improves gRNA detection and annotation in single-cell CRISPR screens.

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Single-cell CRISPR screens in vivo map T cell fate regulomes in cancer.

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Single-cell CRISPR immune screens reveal immunological roles of tumor intrinsic factors.

Project description:Genetic screens are widely exploited to develop novel therapeutic approaches for cancer treatment. With recent advances in single-cell technology, single-cell CRISPR screen (scCRISPR) platforms provide opportunities for target validation and mechanistic studies in a high-throughput manner. Here, we aim to establish scCRISPR platforms which are suitable for immune-related screens involving multiple cell types. We integrated two scCRISPR platforms, namely Perturb-seq and CROP-seq, with both in vitro and in vivo immune screens. By leveraging previously generated resources, we optimized experimental conditions and data analysis pipelines to achieve better consistency between results from high-throughput and individual validations. Furthermore, we evaluated the performance of scCRISPR immune screens in determining underlying mechanisms of tumor intrinsic immune regulation. Our results showed that scCRISPR platforms can simultaneously characterize gene expression profiles and perturbation effects present in individual cells in different immune screen conditions. Results from scCRISPR immune screens also predict transcriptional phenotype associated with clinical responses to cancer immunotherapy. More importantly, scCRISPR screen platforms reveal the interactive relationship between targeting tumor intrinsic factors and T cell-mediated antitumor immune response which cannot be easily assessed by bulk RNA-seq. Collectively, scCRISPR immune screens provide scalable and reliable platforms to elucidate molecular determinants of tumor immune resistance.

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