High-content CRISPR screening

Nature Reviews Methods Primers · 2022 · 563 citations

• Computational biology
• Biology
• Genetics

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

Cell · 2022 · 637 citations

• Biology
• Genetics
• Computational biology

A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (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. 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 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 and cellular function.

Structured elements drive extensive circular RNA translation

Molecular Cell · 2021 · 320 citations

• Biology
• Computational biology
• Genetics

An expanded universe of cancer targets

Cell · 2021 · 219 citations

• Biology
• Computational biology
• Genetics

A community-driven roadmap to advance research on translated open reading frames detected by Ribo-seq

bioRxiv (Cold Spring Harbor Laboratory) · 2021 · 22 citations

• Biology
• Computational biology
• Genetics

ABSTRACT Ribosome profiling (Ribo-seq) has catalyzed a paradigm shift in our understanding of the translational ‘vocabulary’ of the human genome, discovering thousands of translated open reading frames (ORFs) within long non-coding RNAs and presumed untranslated regions of protein-coding genes. However, reference gene annotation projects have been circumspect in their incorporation of these ORFs due to uncertainties about their experimental reproducibility and physiological roles. Yet, it is indisputable that certain Ribo-seq ORFs make stable proteins, others mediate gene regulation, and many have medical implications. Ultimately, the absence of standardized ORF annotation has created a circular problem: while Ribo-seq ORFs remain unannotated by reference biological databases, this lack of characterisation will thwart research efforts examining their roles. Here, we outline the initial stages of a community-led effort supported by GENCODE / Ensembl, HGNC and UniProt to produce a consolidated catalog of human Ribo-seq ORFs.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Cell · 2021 · 784 citations

• Computer Science
• Biology
• Computational biology

While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Single-cell lineages reveal the rates, routes, and drivers of metastasis in cancer xenografts

Science · 2021 · 310 citations

• Cancer research
• Biology
• Oncology

We also show that metastases disseminated via multidirectional tissue routes and complex seeding topologies. Overall, we demonstrate the power of tracing cancer progression at subclonal resolution and vast scale.

Life span extension by glucose restriction is abrogated by methionine supplementation: Cross-talk between glucose and methionine and implication of methionine as a key regulator of life span

Science Advances · 2020 · 71 citations

• Biology
• Cell biology
• Biochemistry

Caloric restriction (CR) is known to extend life span across species; however, the molecular mechanisms are not well understood. We investigate the mechanism by which glucose restriction (GR) extends yeast replicative life span, by combining ribosome profiling and RNA-seq with microfluidic-based single-cell analysis. We discovered a cross-talk between glucose sensing and the regulation of intracellular methionine: GR down-regulated the transcription and translation of methionine biosynthetic enzymes and transporters, leading to a decreased intracellular methionine concentration; external supplementation of methionine cancels the life span extension by GR. Furthermore, genetic perturbations that decrease methionine synthesis/uptake extend life span. These observations suggest that intracellular methionine mediates the life span effects of various nutrient and genetic perturbations, and that the glucose-methionine cross-talk is a general mechanism for coordinating the nutrient status and the translation/growth of a cell. Our work also implicates proteasome as a downstream effector of the life span extension by GR.

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs

Nature Biotechnology · 2020 · 173 citations

• Biology
• Computational biology
• Genetics

Mismatch-CRISPRi Reveals the Co-varying Expression-Fitness Relationships of Essential Genes in Escherichia coli and Bacillus subtilis

Cell Systems · 2020 · 145 citations

• Biology
• Computational biology
• Genetics