Single-cell CRISPR immune screens reveal immunological roles of tumor intrinsic factors
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PROVIDER: PRJNA849218 | ENA |
REPOSITORIES: ENA
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Project description:Genetic screens using CRISPR technology are widely exploited to explore novel targets and discover resistance mechanisms of cancer treatments, including cancer immunotherapy. As it is a time- and resource-intensive manner to individually validate potential candidates and clarify their working mechanisms, single-cell CRISPR screen platforms are designed and developed to parallelly characterize these factors and explore underlying mechanisms in a high-throughput manner. By leveraging the resources generated in our previous studies, Perturb-seq featured with capture sequences integrated into gRNA-expressing vectors and CROP-seq featured with poly(A)-tailed gRNA transcripts under the control of RNAPII promoters were selected among several independent single-cell CRISPR screen platforms to directly evaluate the performance of different scCRISPR screen platforms in determining underlying mechanisms of tumor intrinsic immune regulation and optimize experimental conditions and data analysis in immune-related scCRISPR screens. In the study, the influence of each perturbation on the gene expression profile and the response to in vitro T cell killing and in vivo anti-PD-1 treatment were comprehensively appraised at the single-cell level. It is clearly demonstrated that scCRISPR screen platforms could efficiently characterize gene expression profiles and perturbation effects present in individual cells during both in vitro and in vivo immune-related screen processes. Particularly, scCRISPR screen platforms presented more detail to determine heterogeneous change of gene expression in distinct knockout tumor cells in the presence of immune attack which cannot be easily assessed by canonical bulk RNA-seq. Collectively, scCRISPR screen technologies provide scalable and reliable platforms to validate identified tumor intrinsic immune factors identified from genome-wide CRISPR screens and elucidate their potential working mechanisms.
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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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