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

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.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes.

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.

Mapping the genetic landscape of DNA double-strand break repair.

Cells repair DNA double-strand breaks (DSBs) through a complex set of pathways critical for maintaining genomic integrity. To systematically map these pathways, we developed a high-throughput screening approach called Repair-seq that measures the effects of thousands of genetic perturbations on mutations introduced at targeted DNA lesions. Using Repair-seq, we profiled DSB repair products induced by two programmable nucleases (Cas9 and Cas12a) in the presence or absence of oligonucleotides for homology-directed repair (HDR) after knockdown of 476 genes involved in DSB repair or associated processes. The resulting data enabled principled, data-driven inference of DSB end joining and HDR pathways. Systematic interrogation of this data uncovered unexpected relationships among DSB repair genes and demonstrated that repair outcomes with superficially similar sequence architectures can have markedly different genetic dependencies. This work provides a foundation for mapping DNA repair pathways and for optimizing genome editing across diverse modalities.

Deciphering cell states and genealogies of human haematopoiesis.

The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)1. Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems2-5, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.

GIGYF2 and 4EHP Inhibit Translation Initiation of Defective Messenger RNAs to Assist Ribosome-Associated Quality Control.

Ribosome-associated quality control (RQC) pathways protect cells from toxicity caused by incomplete protein products resulting from translation of damaged or problematic mRNAs. Extensive work in yeast has identified highly conserved mechanisms that lead to degradation of faulty mRNA and partially synthesized polypeptides. Here we used CRISPR-Cas9-based screening to search for additional RQC strategies in mammals. We found that failed translation leads to specific inhibition of translation initiation on that message. This negative feedback loop is mediated by two translation inhibitors, GIGYF2 and 4EHP. Model substrates and growth-based assays established that inhibition of additional rounds of translation acts in concert with known RQC pathways to prevent buildup of toxic proteins. Inability to block translation of faulty mRNAs and subsequent accumulation of partially synthesized polypeptides could explain the neurodevelopmental and neuropsychiatric disorders observed in mice and humans with compromised GIGYF2 function.

Understanding Biases in Ribosome Profiling Experiments Reveals Signatures of Translation Dynamics in Yeast.

Ribosome profiling produces snapshots of the locations of actively translating ribosomes on messenger RNAs. These snapshots can be used to make inferences about translation dynamics. Recent ribosome profiling studies in yeast, however, have reached contradictory conclusions regarding the average translation rate of each codon. Some experiments have used cycloheximide (CHX) to stabilize ribosomes before measuring their positions, and these studies all counterintuitively report a weak negative correlation between the translation rate of a codon and the abundance of its cognate tRNA. In contrast, some experiments performed without CHX report strong positive correlations. To explain this contradiction, we identify unexpected patterns in ribosome density downstream of each type of codon in experiments that use CHX. These patterns are evidence that elongation continues to occur in the presence of CHX but with dramatically altered codon-specific elongation rates. The measured positions of ribosomes in these experiments therefore do not reflect the amounts of time ribosomes spend at each position in vivo. These results suggest that conclusions from experiments in yeast using CHX may need reexamination. In particular, we show that in all such experiments, codons decoded by less abundant tRNAs were in fact being translated more slowly before the addition of CHX disrupted these dynamics.

High-throughput DNA sequencing errors are reduced by orders of magnitude using circle sequencing.

A major limitation of high-throughput DNA sequencing is the high rate of erroneous base calls produced. For instance, Illumina sequencing machines produce errors at a rate of ~0.1-1 × 10(-2) per base sequenced. These technologies typically produce billions of base calls per experiment, translating to millions of errors. We have developed a unique library preparation strategy, "circle sequencing," which allows for robust downstream computational correction of these errors. In this strategy, DNA templates are circularized, copied multiple times in tandem with a rolling circle polymerase, and then sequenced on any high-throughput sequencing machine. Each read produced is computationally processed to obtain a consensus sequence of all linked copies of the original molecule. Physically linking the copies ensures that each copy is independently derived from the original molecule and allows for efficient formation of consensus sequences. The circle-sequencing protocol precedes standard library preparations and is therefore suitable for a broad range of sequencing applications. We tested our method using the Illumina MiSeq platform and obtained errors in our processed sequencing reads at a rate as low as 7.6 × 10(-6) per base sequenced, dramatically improving the error rate of Illumina sequencing and putting error on par with low-throughput, but highly accurate, Sanger sequencing. Circle sequencing also had substantially higher efficiency and lower cost than existing barcode-based schemes for correcting sequencing errors.

CRISPR-based functional genomics in human dendritic cells.

Dendritic cells (DCs) regulate processes ranging from antitumor and antiviral immunity to host-microbe communication at mucosal surfaces. It remains difficult, however, to genetically manipulate human DCs, limiting our ability to probe how DCs elicit specific immune responses. Here, we develop a CRISPR-Cas9 genome editing method for human monocyte-derived DCs (moDCs) that mediates knockouts with a median efficiency of >94% across >300 genes. Using this method, we perform genetic screens in moDCs, identifying mechanisms by which DCs tune responses to lipopolysaccharides from the human microbiome. In addition, we reveal donor-specific responses to lipopolysaccharides, underscoring the importance of assessing immune phenotypes in donor-derived cells, and identify candidate genes that control this specificity, highlighting the potential of our method to pinpoint determinants of inter-individual variation in immunity. Our work sets the stage for a systematic dissection of the immune signaling at the host-microbiome interface and for targeted engineering of DCs for neoantigen vaccination.

Regulation of translation by methylation multiplicity of 18S rRNA.

N6-methyladenosine (m6A) is a conserved ribonucleoside modification that regulates many facets of RNA metabolism. Using quantitative mass spectrometry, we find that the universally conserved tandem adenosines at the 3' end of 18S rRNA, thought to be constitutively di-methylated (m62A), are also mono-methylated (m6A). Although present at substoichiometric amounts, m6A at these positions increases significantly in response to sulfur starvation in yeast cells and mammalian cell lines. Combining yeast genetics and ribosome profiling, we provide evidence to suggest that m6A-bearing ribosomes carry out translation distinctly from m62A-bearing ribosomes, featuring a striking specificity for sulfur metabolism genes. Our work thus reveals methylation multiplicity as a mechanism to regulate translation.

Efficient C•G-to-G•C base editors developed using CRISPRi screens, target-library analysis, and machine learning.

Programmable C•G-to-G•C base editors (CGBEs) have broad scientific and therapeutic potential, but their editing outcomes have proved difficult to predict and their editing efficiency and product purity are often low. We describe a suite of engineered CGBEs paired with machine learning models to enable efficient, high-purity C•G-to-G•C base editing. We performed a CRISPR interference (CRISPRi) screen targeting DNA repair genes to identify factors that affect C•G-to-G•C editing outcomes and used these insights to develop CGBEs with diverse editing profiles. We characterized ten promising CGBEs on a library of 10,638 genomically integrated target sites in mammalian cells and trained machine learning models that accurately predict the purity and yield of editing outcomes (R = 0.90) using these data. These CGBEs enable correction to the wild-type coding sequence of 546 disease-related transversion single-nucleotide variants (SNVs) with >90% precision (mean 96%) and up to 70% efficiency (mean 14%). Computational prediction of optimal CGBE-single-guide RNA pairs enables high-purity transversion base editing at over fourfold more target sites than achieved using any single CGBE variant.

Inference of single-cell phylogenies from lineage tracing data using Cassiopeia.

The pairing of CRISPR/Cas9-based gene editing with massively parallel single-cell readouts now enables large-scale lineage tracing. However, the rapid growth in complexity of data from these assays has outpaced our ability to accurately infer phylogenetic relationships. First, we introduce Cassiopeia-a suite of scalable maximum parsimony approaches for tree reconstruction. Second, we provide a simulation framework for evaluating algorithms and exploring lineage tracer design principles. Finally, we generate the most complex experimental lineage tracing dataset to date, 34,557 human cells continuously traced over 15 generations, and use it for benchmarking phylogenetic inference approaches. We show that Cassiopeia outperforms traditional methods by several metrics and under a wide variety of parameter regimes, and provide insight into the principles for the design of improved Cas9-enabled recorders. Together, these should broadly enable large-scale mammalian lineage tracing efforts. Cassiopeia and its benchmarking resources are publicly available at www.github.com/YosefLab/Cassiopeia.

Titrating gene expression using libraries of systematically attenuated CRISPR guide RNAs.

A lack of tools to precisely control gene expression has limited our ability to evaluate relationships between expression levels and phenotypes. Here, we describe an approach to titrate expression of human genes using CRISPR interference and series of single-guide RNAs (sgRNAs) with systematically modulated activities. We used large-scale measurements across multiple cell models to characterize activities of sgRNAs containing mismatches to their target sites and derived rules governing mismatched sgRNA activity using deep learning. These rules enabled us to synthesize a compact sgRNA library to titrate expression of ~2,400 genes essential for robust cell growth and to construct an in silico sgRNA library spanning the human genome. Staging cells along a continuum of gene expression levels combined with single-cell RNA-seq readout revealed sharp transitions in cellular behaviors at gene-specific expression thresholds. Our work provides a general tool to control gene expression, with applications ranging from tuning biochemical pathways to identifying suppressors for diseases of dysregulated gene expression.

Combinatorial single-cell CRISPR screens by direct guide RNA capture and targeted sequencing.

Single-cell CRISPR screens enable the exploration of mammalian gene function and genetic regulatory networks. However, use of this technology has been limited by reliance on indirect indexing of single-guide RNAs (sgRNAs). Here we present direct-capture Perturb-seq, a versatile screening approach in which expressed sgRNAs are sequenced alongside single-cell transcriptomes. Direct-capture Perturb-seq enables detection of multiple distinct sgRNA sequences from individual cells and thus allows pooled single-cell CRISPR screens to be easily paired with combinatorial perturbation libraries that contain dual-guide expression vectors. We demonstrate the utility of this approach for high-throughput investigations of genetic interactions and, leveraging this ability, dissect epistatic interactions between cholesterol biogenesis and DNA repair. Using direct capture Perturb-seq, we also show that targeting individual genes with multiple sgRNAs per cell improves efficacy of CRISPR interference and activation, facilitating the use of compact, highly active CRISPR libraries for single-cell screens. Last, we show that hybridization-based target enrichment permits sensitive, specific sequencing of informative transcripts from single-cell RNA-seq experiments.

Ribosome Profiling: Global Views of Translation.

The translation of messenger RNA (mRNA) into protein and the folding of the resulting protein into an active form are prerequisites for virtually every cellular process and represent the single largest investment of energy by cells. Ribosome profiling-based approaches have revolutionized our ability to monitor every step of protein synthesis in vivo, allowing one to measure the rate of protein synthesis across the proteome, annotate the protein coding capacity of genomes, monitor localized protein synthesis, and explore cotranslational folding and targeting. The rich and quantitative nature of ribosome profiling data provides an unprecedented opportunity to explore and model complex cellular processes. New analytical techniques and improved experimental protocols will provide a deeper understanding of the factors controlling translation speed and its impact on protein function and cell physiology as well as the role of ribosomal RNA and mRNA modifications in regulating translation.

The ER membrane protein complex interacts cotranslationally to enable biogenesis of multipass membrane proteins.

The endoplasmic reticulum (ER) supports biosynthesis of proteins with diverse transmembrane domain (TMD) lengths and hydrophobicity. Features in transmembrane domains such as charged residues in ion channels are often functionally important, but could pose a challenge during cotranslational membrane insertion and folding. Our systematic proteomic approaches in both yeast and human cells revealed that the ER membrane protein complex (EMC) binds to and promotes the biogenesis of a range of multipass transmembrane proteins, with a particular enrichment for transporters. Proximity-specific ribosome profiling demonstrates that the EMC engages clients cotranslationally and immediately following clusters of TMDs enriched for charged residues. The EMC can remain associated after completion of translation, which both protects clients from premature degradation and allows recruitment of substrate-specific and general chaperones. Thus, the EMC broadly enables the biogenesis of multipass transmembrane proteins containing destabilizing features, thereby mitigating the trade-off between function and stability.

Ribosomal Architecture: Constraints Imposed by the Need for Self-Production.

Ribosomes contain proteins that must themselves be made by ribosomes. A new study shows that splitting ribosomal protein content into many small, similarly sized units maximizes the efficiency of this synthesis, suggesting that ribosomal architecture has been shaped by evolutionary pressure to efficiently self-synthesize.

CAT-tailing as a fail-safe mechanism for efficient degradation of stalled nascent polypeptides.

Ribosome stalling leads to recruitment of the ribosome quality control complex (RQC), which targets the partially synthesized polypeptide for proteasomal degradation through the action of the ubiquitin ligase Ltn1p. A second core RQC component, Rqc2p, modifies the nascent polypeptide by adding a carboxyl-terminal alanine and threonine (CAT) tail through a noncanonical elongation reaction. Here we examined the role of CAT-tailing in nascent-chain degradation in budding yeast. We found that Ltn1p efficiently accessed only nascent-chain lysines immediately proximal to the ribosome exit tunnel. For substrates without Ltn1p-accessible lysines, CAT-tailing enabled degradation by exposing lysines sequestered in the ribosome exit tunnel. Thus, CAT-tails do not serve as a degron, but rather provide a fail-safe mechanism that expands the range of RQC-degradable substrates.

Improved Ribosome-Footprint and mRNA Measurements Provide Insights into Dynamics and Regulation of Yeast Translation.

Ribosome-footprint profiling provides genome-wide snapshots of translation, but technical challenges can confound its analysis. Here, we use improved methods to obtain ribosome-footprint profiles and mRNA abundances that more faithfully reflect gene expression in Saccharomyces cerevisiae. Our results support proposals that both the beginning of coding regions and codons matching rare tRNAs are more slowly translated. They also indicate that emergent polypeptides with as few as three basic residues within a ten-residue window tend to slow translation. With the improved mRNA measurements, the variation attributable to translational control in exponentially growing yeast was less than previously reported, and most of this variation could be predicted with a simple model that considered mRNA abundance, upstream open reading frames, cap-proximal structure and nucleotide composition, and lengths of the coding and 5' UTRs. Collectively, our results provide a framework for executing and interpreting ribosome-profiling studies and reveal key features of translational control in yeast.