CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue.

Autor:	Gu J; Department of Biological Sciences, Columbia University, NY, USA., Iyer A; Department of Biological Sciences, Columbia University, NY, USA., Wesley B; Department of Biological Sciences, Columbia University, NY, USA., Taglialatela A; Department of Genetics and Development, Columbia University Irving Medical Center, NY, USA., Leuzzi G; Department of Genetics and Development, Columbia University Irving Medical Center, NY, USA.; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA.; Institute for Cancer Genetics, Columbia University Irving Medical Center, NY, USA., Hangai S; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA., Decker A; Department of Biological Sciences, Columbia University, NY, USA., Gu R; Department of Biological Sciences, Columbia University, NY, USA., Klickstein N; Department of Biological Sciences, Columbia University, NY, USA., Shuai Y; Department of Biological Sciences, Columbia University, NY, USA., Jankovic K; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA., Parker-Burns L; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA., Jin Y; Department of Biomedical Engineering, Columbia University, NY, USA., Zhang JY; Department of Biomedical Engineering, Columbia University, NY, USA., Hong J; Department of Computer Science, Columbia University, NY, USA., Niu S; Weill Cornell Medicine, NY, USA.; Genentech Research and Early Development, CA, USA., Chou J; Department of Biological Sciences, Columbia University, NY, USA.; Weill Cornell Medicine, NY, USA., Landau DA; Weill Cornell Medicine, NY, USA.; New York Genome Center, NY, USA., Azizi E; Department of Biomedical Engineering, Columbia University, NY, USA.; Department of Computer Science, Columbia University, NY, USA.; Irving Institute for Cancer Dynamics, Columbia University, NY, USA., Chan EM; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA.; New York Genome Center, NY, USA.; Department of Medicine, Division of Hematology/Oncology, Columbia University Irving Medical Center, NY, USA., Ciccia A; Department of Genetics and Development, Columbia University Irving Medical Center, NY, USA.; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA.; Institute for Cancer Genetics, Columbia University Irving Medical Center, NY, USA., Gaublomme JT; Department of Biological Sciences, Columbia University, NY, USA.; Herbert Irving Comprehensive Cancer Center, Columbia University Irving Medical Center, NY, USA.; New York Genome Center, NY, USA.; Irving Institute for Cancer Dynamics, Columbia University, NY, USA.
Jazyk:	angličtina
Zdroj:	BioRxiv : the preprint server for biology [bioRxiv] 2023 Dec 26. Date of Electronic Publication: 2023 Dec 26.
DOI:	10.1101/2023.12.26.572587
Abstrakt:	Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease. Competing Interests: Declaration of competing interests Columbia University has filed a patent application related to this work for which J.T.G. is an inventor. E.M.C.’s laboratory receives support from Novartis. D.A.L. has served as a consultant for AbbVie, AstraZeneca, and Illumina and is on the Scientific Advisory Board of Mission Bio, Pangea, Alethiomics, and C2i Genomics; D.A.L. has received prior research funding from BMS, 10x Genomics, Ultima Genomics, and Illumina unrelated to the current manuscript.
Databáze:	MEDLINE
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