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CRISPR screens for functional interrogation of immunity

Abstract

CRISPR-based technologies represent a major breakthrough in biomedical science as they offer a powerful platform for unbiased screening and functional genomics in various fields, including immunology. Pooled and arrayed CRISPR screens have uncovered previously unknown intracellular drivers in innate and adaptive immune cells for immune regulation as well as intercellular regulators mediating cell–cell interactions. Recent single-cell CRISPR screening platforms expand the readouts to the transcriptome and enable the inference of gene regulatory networks for better mechanistic insights. CRISPR screens also allow for mapping of genetic interactions to identify genes that synergize or alleviate complex immune phenotypes. Here, we review the progress in and emerging adaptation of CRISPR technologies to advance our fundamental immunological knowledge and identify novel disease targets for immunotherapy of infection, inflammation and cancer.

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Fig. 1: In vitro CRISPR screens for immune cell activation and function.
Fig. 2: In vivo CRISPR screens in immune cells and tumour cells for regulators of the immune response.
Fig. 3: Integration of gene regulatory network analysis with CRISPR screens.
Fig. 4: CRISPR-based genetic interaction screens in immune regulation.

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Acknowledgements

The authors acknowledge all investigators whose contributions could not be discussed owing to space limitations, and N. Chapman for critical reading and editing of the manuscript. Research in the Chi laboratory was supported by ALSAC, US National Institutes of Health AI105887, AI131703, AI140761, AI150241, AI150514 and CA253188.

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All authors contributed to discussion of content for the article and to writing and editing of the manuscript.

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Correspondence to Hongbo Chi.

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Competing interests

J.G.D. consults for Microsoft Research, Abata Therapeutics, Servier, Maze Therapeutics, BioNTech, Sangamo and Pfizer; consults for and has equity in Tango Therapeutics; serves as a paid scientific adviser to the Laboratory for Genomics Research, funded in part by GlaxoSmithKline; and receives funding support from the Functional Genomics Consortium: Abbvie, Bristol Myers Squibb, Janssen, Merck and Vir Biotechnology. Interests of J.G.D. were reviewed and are managed by the Broad Institute in accordance with its conflict of interest policies. H.C. consults for Kumquat Biosciences. H.S. declares no competing interests.

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The Cancer Genome Atlas: https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga

Supplementary information

Glossary

Base editing

A CRISPR–Cas9-based genome editing technology that introduces point mutations in DNA without generating double-strand breaks by tethering a nuclease-defective Cas9-D10A nickase variant (Cas9n) to a deaminase.

Coverage

The average number of cells perturbed by each sgRNA in a pooled CRISPR screen, with a coverage of 500 cells generally recommended for screens to achieve optimal signal to noise ratio, although a lower coverage (such as 200 cells) may be used for enrichment screens.

CRISPR-mediated genetically engineered mouse models

(CRISPR-GEMMs). Mouse models enabled by CRISPR technology for pooled targeting of multiple genes through somatic genome editing and large-scale direct in vivo screening.

CRISPR-sciATAC

A method that simultaneously captures transcripts encoding an sgRNA and carries out single-cell combinatorial indexing ATAC-seq based on a unique combination of barcodes, which tag both the sgRNA and ATAC fragments from each cell.

CRISP-seq

A platform that integrates the resolution of massively parallel scRNA-seq with the genome editing scale of pooled CRISPR screens, with sgRNA identification inferred by a transcribed poly-adenylated unique guide index on the same vector.

CROP-seq

This platform makes use of an sgRNA delivery vector system that duplicates the sequence of a single-encoded sgRNA during lentiviral transduction to produce two expression cassettes on the same construct. One cassette expresses a functional sgRNA and the other expresses a polyadenylated transcript carrying the sgRNA sequence at the 3′ end for detection of individual sgRNAs in droplet-based single-cell RNA sequencing.

ECCITE-seq

Expanded CRISPR-compatible CITE-seq, which enables 5′ capture-based scRNA-seq, T cell receptor or B cell receptor V(D)J reconstruction, and surface protein marker detection as readouts, together with single-cell sgRNA sequence capture.

Mosaic-seq

Mosaic single-cell analysis by indexed CRISPR sequencing, a method that jointly measures the transcriptome of a cell and its sgRNA modulators inferred from the barcode sequences on the transduced sgRNA lentiviral vector backbone.

Perturb-ATAC

A method that combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling (assay for transposase accessible chromatin (ATAC)) in single cells captured by the Integrated Fluidics Circuit (Fluidigm) chambers.

Perturb-CITE-seq

A combined platform of 3′ droplet-based scRNA-seq with extracellular protein detection (by CITE-seq, which uses DNA-barcoded antibodies to convert the detection of proteins into a quantitative readout) and single-cell sgRNA detection. The method expresses an sgRNA on a polyadenylated transcript using a modified CROP-seq vector and performs a targeted ‘dial-out’ PCR amplification, which robustly links sgRNA identities to single-cell transcriptional and protein profiles.

Perturb-seq

A platform that combines a pooled CRISPR screen with single-cell RNA sequencing (scRNA-seq) by encoding the identity of the perturbation on an expressed guide barcode.

Prime editing

A CRISPR–Cas9-based genome editing technology that introduces new sequence information into the genome by fusing Cas9-H840A nickase to a reverse transcriptase enzyme that promotes genome modification via a sequence template encoded within an extended prime editing guide RNA.

Pro-Codes

A protein barcoding vector system with combinatorial arrangements of linear epitopes, each paired with a different CRISPR sgRNA, for the analysis of multiple proteins to identify cells expressing different CRISPR sgRNAs at single-cell resolution.

Pseudotime

The ordering of each cell along a developmental lineage based on gene expression as profiled by scRNA-seq.

Single-guide RNA

(sgRNA). A single RNA molecule used to direct Cas9 protein to bind and cleave a particular DNA sequence for genome editing.

Spear-ATAC

A platform that enables simultaneous readouts of chromatin accessibility profiles and integrated sgRNA from thousands of individual cells by reading out sgRNA spacer sequences directly from genomic DNA rather than from RNA transcripts.

Tiling sgRNA library

An sgRNA library that is designed to incorporate many editing sites across the length of a gene and its regulatory elements to comprehensively evaluate their associated phenotypes.

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Shi, H., Doench, J.G. & Chi, H. CRISPR screens for functional interrogation of immunity. Nat Rev Immunol (2022). https://doi.org/10.1038/s41577-022-00802-4

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