A new era in functional genomics screens

Przybyla, Laralynne; Gilbert, Luke A.

doi:10.1038/s41576-021-00409-w

Cited by 156 publications

(123 citation statements)

References 222 publications

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“…Pooled forward genetic screens typically use low-dimensional phenotypes (e.g., growth, marker gene expression, drug resistance) for selection. The use of simple phenotypes can conflate genes acting via different mechanisms, requiring extensive follow-up studies to disentangle genetic pathways ( 8 ). Additionally, in forward genetics, serendipitous discovery is constrained by the prerequisite of selecting phenotypes prior to screening.…”

Section: Main Textmentioning

confidence: 99%

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

Replogle¹,

Saunders²,

Pogson³

et al. 2021

Preprint

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A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based screens with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, to date, have been used at limited scales. Here, we perform genome-scale Perturb-seq targeting all expressed genes with CRISPR interference (CRISPRi) across >2.5 million human cells and present a framework to power biological discovery with the resulting genotype-phenotype map. We use transcriptional phenotypes to predict the function of poorly-characterized genes, uncovering new regulators of ribosome biogenesis (including CCDC86, ZNF236, and SPATA5L1), transcription (C7orf26), and mitochondrial respiration (TMEM242). In addition to assigning gene function, single-cell transcriptional phenotypes allow for in-depth dissection of complex cellular phenomena – from RNA processing to differentiation. We leverage this ability to systematically identify the genetic drivers and consequences of aneuploidy and to discover an unanticipated layer of stress-specific regulation of the mitochondrial genome. Our information-rich genotype-phenotype map reveals a multidimensional portrait of gene function and cellular behavior.

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Section: Main Textmentioning

confidence: 99%

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

Replogle¹,

Saunders²,

Pogson³

et al. 2021

Preprint

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“…Pooled screening with CRISPR technology has revolutionized the ease and scale for probing gene function 1,2 . Targeted loci, which are often protein coding genes, can each have hundreds of protospacer adjacent motifs (PAMs), providing many potential single guide RNA (sgRNA) options.…”

Section: Introductionmentioning

confidence: 99%

Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening

DeWeirdt

McGee

Zheng

et al. 2022

Preprint

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CRISPR technology is a powerful tool for studying genome function. To aid in picking sgRNAs that have maximal efficacy against a target of interest from many possible options, several groups have developed models that predict sgRNA on-target activity. Although multiple tracrRNA variants are commonly used for screening, no existing models account for this feature when nominating sgRNAs. Here we develop an on-target model, Rule Set 3, that makes optimal predictions for multiple tracrRNA variants. We validate Rule Set 3 on a new dataset of sgRNAs tiling essential and non-essential genes, demonstrating substantial improvement over prior prediction models. By analyzing the differences in sgRNA activity between tracrRNA variants, we show that Pol III transcription termination is a strong determinant of sgRNA activity. We expect these results to improve the performance of CRISPR screening and inform future research on tracrRNA engineering and sgRNA modeling.

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“…These hypotheses then need to be experimentally validated. Although still somewhat limited by effect size, efficiency and implementation in disease relevant primary cells, clustered regularly interspaced short palindromic repeats (CRISPR)-based methods have become the gold standard for the functional dissection of GWAS loci in recent years [103]. The use of CRISPR-based technologies and massively parallel reporter assays (MPRAs) to functionally interpret GWAS signals is reviewed extensively elsewhere in this issue of Seminars in Immunopathology [104].…”

Section: Discussionmentioning

confidence: 99%

Fine mapping with epigenetic information and 3D structure

Orozco

2022

Semin Immunopathol

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Since 2005, thousands of genome-wide association studies (GWAS) have been published, identifying hundreds of thousands of genetic variants that increase risk of complex traits such as autoimmune diseases. This wealth of data has the potential to improve patient care, through personalized medicine and the identification of novel drug targets. However, the potential of GWAS for clinical translation has not been fully achieved yet, due to the fact that the functional interpretation of risk variants and the identification of causal variants and genes are challenging. The past decade has seen the development of great advances that are facilitating the overcoming of these limitations, by utilizing a plethora of genomics and epigenomics tools to map and characterize regulatory elements and chromatin interactions, which can be used to fine map GWAS loci, and advance our understanding of the biological mechanisms that cause disease.

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A new era in functional genomics screens

Cited by 156 publications

References 222 publications

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq

Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening

Fine mapping with epigenetic information and 3D structure

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