The PRC2.1 Subcomplex Opposes G1 Progression through Regulation of CCND1 and CCND2.

Progression through the G1 phase of the cell cycle is the most highly regulated step in cellular division. We employed a chemogenomics approach to discover novel cellular networks that regulate cell cycle progression. This approach uncovered functional clusters of genes that altered sensitivity of cells to inhibitors of the G1/S transition. Mutation of components of the Polycomb Repressor Complex 2 rescued growth inhibition caused by the CDK4/6 inhibitor palbociclib, but not to inhibitors of S phase or mitosis. In addition to its core catalytic subunits, mutation of the PRC2.1 accessory protein MTF2, but not the PRC2.2 protein JARID2, rendered cells resistant to palbociclib treatment. We found that PRC2.1 (MTF2), but not PRC2.2 (JARID2), was critical for promoting H3K27me3 deposition at CpG islands genome-wide and in promoters. This included the CpG islands in the promoter of the CDK4/6 cyclins CCND1 and CCND2, and loss of MTF2 lead to upregulation of both CCND1 and CCND2. Our results demonstrate a role for PRC2.1, but not PRC2.2, in promoting G1 progression.

A scalable platform for efficient CRISPR-Cas9 chemical-genetic screens of DNA damage-inducing compounds.

Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screening platform by generating a DNA damage response (DDR)-focused custom sgRNA library targeting 1011 genes with 3033 sgRNAs. We performed five proof-of-principle compound screens and found that the compounds' known modes-of-action (MoA) were enriched among the compounds' CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale with 20-fold fewer resources than commonly used genome-wide libraries and produce biologically informative CGI profiles.

Dimensionality reduction methods for extracting functional networks from large-scale CRISPR screens.

CRISPR-Cas9 screens facilitate the discovery of gene functional relationships and phenotype-specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole-genome CRISPR screens aimed at identifying cancer-specific genetic dependencies across human cell lines. A mitochondria-associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co-essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods-autoencoders, robust, and classical principal component analyses (PCA)-for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low-dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction-based normalization tools.

RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.

DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.

Reproducibility metrics for context-specific CRISPR screens.

CRISPR screens are used extensively to systematically interrogate the phenotype-to-genotype problem. In contrast to early CRISPR screens, which defined core cell fitness genes, most current efforts now aim to identify context-specific phenotypes that differentiate a cell line, genetic background, or condition of interest, such as a drug treatment. While CRISPR-related technologies have shown great promise and a fast pace of innovation, a better understanding of standards and methods for quality assessment of CRISPR screen results is crucial to guide technology development and application. Specifically, many commonly used metrics for quantifying screen quality do not accurately measure the reproducibility of context-specific hits. We highlight the importance of reporting reproducibility statistics that directly relate to the purpose of the screen and suggest the use of metrics that are sensitive to context-specific signal. A record of this paper's transparent peer review process is included in the supplemental information.

Dimensionality reduction methods for extracting functional networks from large-scale CRISPR screens.

CRISPR-Cas9 screens facilitate the discovery of gene functional relationships and phenotype-specific dependencies. The Cancer Dependency Map (DepMap) is the largest compendium of whole-genome CRISPR screens aimed at identifying cancer-specific genetic dependencies across human cell lines. A mitochondria-associated bias has been previously reported to mask signals for genes involved in other functions, and thus, methods for normalizing this dominant signal to improve co-essentiality networks are of interest. In this study, we explore three unsupervised dimensionality reduction methods - autoencoders, robust, and classical principal component analyses (PCA) - for normalizing the DepMap to improve functional networks extracted from these data. We propose a novel "onion" normalization technique to combine several normalized data layers into a single network. Benchmarking analyses reveal that robust PCA combined with onion normalization outperforms existing methods for normalizing the DepMap. Our work demonstrates the value of removing low-dimensional signals from the DepMap before constructing functional gene networks and provides generalizable dimensionality reduction-based normalization tools.

Survival-based CRISPR genetic screens across a panel of permissive cell lines identify common and cell-specific SARS-CoV-2 host factors.

SARS-CoV-2 depends on host cell components for infection and replication. Identification of virus-host dependencies offers an effective way to elucidate mechanisms involved in viral infection and replication. If druggable, host factor dependencies may present an attractive strategy for anti-viral therapy. In this study, we performed genome wide CRISPR knockout screens in Vero E6 cells and four human cell lines including Calu-3, UM-UC-4, HEK-293 and HuH-7 to identify genetic regulators of SARS-CoV-2 infection. Our findings identified only ACE2, the cognate SARS-CoV-2 entry receptor, as a common host dependency factor across all cell lines, while other host genes identified were largely cell line specific, including known factors TMPRSS2 and CTSL. Several of the discovered host-dependency factors converged on pathways involved in cell signalling, immune-related pathways, and chromatin modification. Notably, the chromatin modifier gene KMT2C in Calu-3 cells had the strongest impact in preventing SARS-CoV-2 infection when perturbed.

Analysis of combinatorial CRISPR screens with the Orthrus scoring pipeline.

The continued improvement of combinatorial CRISPR screening platforms necessitates the development of new computational pipelines for scoring combinatorial screening data. Unlike for single-guide RNA (sgRNA) pooled screening platforms, combinatorial scoring for multiplexed systems is confounded by guide design parameters such as the number of gRNAs per construct, the position of gRNAs along constructs, and additional features that may impact gRNA expression, processing or capture. In this protocol we describe Orthrus, an R package for processing, scoring and analyzing combinatorial CRISPR screening data that addresses these challenges. This protocol walks through the application of Orthrus to previously published combinatorial screening data from the CHyMErA experimental system, a platform we recently developed that pairs Cas9 with Cas12a gRNAs and enables programmed targeting of multiple genomic sites. We demonstrate Orthrus' features for screen quality assessment and two distinct scoring modes for dual guide RNAs (dgRNAs) that target the same gene twice or dgRNAs that target two different genes. Running Orthrus requires basic R programming experience, ~5-10 min of computational time and 15-60 min total.

A method for benchmarking genetic screens reveals a predominant mitochondrial bias.

We present FLEX (Functional evaluation of experimental perturbations), a pipeline that leverages several functional annotation resources to establish reference standards for benchmarking human genome-wide CRISPR screen data and methods for analyzing them. FLEX provides a quantitative measurement of the functional information captured by a given gene-pair dataset and a means to explore the diversity of functions captured by the input dataset. We apply FLEX to analyze data from the diverse cell line screens generated by the DepMap project. We identify a predominant mitochondria-associated signal within co-essentiality networks derived from these data and explore the basis of this signal. Our analysis and time-resolved CRISPR screens in a single cell line suggest that the variable phenotypes associated with mitochondria genes across cells may reflect screen dynamics and protein stability effects rather than genetic dependencies. We characterize this functional bias and demonstrate its relevance for interpreting differential hits in any CRISPR screening context. More generally, we demonstrate the utility of the FLEX pipeline for performing robust comparative evaluations of CRISPR screens or methods for processing them.

Genetic interaction mapping and exon-resolution functional genomics with a hybrid Cas9-Cas12a platform.

Systematic mapping of genetic interactions (GIs) and interrogation of the functions of sizable genomic segments in mammalian cells represent important goals of biomedical research. To advance these goals, we present a CRISPR (clustered regularly interspaced short palindromic repeats)-based screening system for combinatorial genetic manipulation that employs coexpression of CRISPR-associated nucleases 9 and 12a (Cas9 and Cas12a) and machine-learning-optimized libraries of hybrid Cas9-Cas12a guide RNAs. This system, named Cas Hybrid for Multiplexed Editing and screening Applications (CHyMErA), outperforms genetic screens using Cas9 or Cas12a editing alone. Application of CHyMErA to the ablation of mammalian paralog gene pairs reveals extensive GIs and uncovers phenotypes normally masked by functional redundancy. Application of CHyMErA in a chemogenetic interaction screen identifies genes that impact cell growth in response to mTOR pathway inhibition. Moreover, by systematically targeting thousands of alternative splicing events, CHyMErA identifies exons underlying human cell line fitness. CHyMErA thus represents an effective screening approach for GI mapping and the functional analysis of sizable genomic regions, such as alternative exons.

MaGIC: a machine learning tool set and web application for monoallelic gene inference from chromatin.

BACKGROUND: A large fraction of human and mouse autosomal genes are subject to random monoallelic expression (MAE), an epigenetic mechanism characterized by allele-specific gene expression that varies between clonal cell lineages. MAE is highly cell-type specific and mapping it in a large number of cell and tissue types can provide insight into its biological function. Its detection, however, remains challenging. RESULTS: We previously reported that a sequence-independent chromatin signature identifies, with high sensitivity and specificity, genes subject to MAE in multiple tissue types using readily available ChIP-seq data. Here we present an implementation of this method as a user-friendly, open-source software pipeline for monoallelic gene inference from chromatin (MaGIC). The source code for the MaGIC pipeline and the Shiny app is available at https://github.com/gimelbrantlab/magic . CONCLUSION: The pipeline can be used by researchers to map monoallelic expression in a variety of cell types using existing models and to train new models with additional sets of chromatin marks.

Using BEAN-counter to quantify genetic interactions from multiplexed barcode sequencing experiments.

The construction of genome-wide mutant collections has enabled high-throughput, high-dimensional quantitative characterization of gene and chemical function, particularly via genetic and chemical-genetic interaction experiments. As the throughput of such experiments increases with improvements in sequencing technology and sample multiplexing, appropriate tools must be developed to handle the large volume of data produced. Here, we describe how to apply our approach to high-throughput, fitness-based profiling of pooled mutant yeast collections using the BEAN-counter software pipeline (Barcoded Experiment Analysis for Next-generation sequencing) for analysis. The software has also successfully processed data from Schizosaccharomyces pombe, Escherichia coli, and Zymomonas mobilis mutant collections. We provide general recommendations for the design of large-scale, multiplexed barcode sequencing experiments. The procedure outlined here was used to score interactions for ~4 million chemical-by-mutant combinations in our recently published chemical-genetic interaction screen of nearly 14,000 chemical compounds across seven diverse compound collections. Here we selected a representative subset of these data on which to demonstrate our analysis pipeline. BEAN-counter is open source, written in Python, and freely available for academic use. Users should be proficient at the command line; advanced users who wish to analyze larger datasets with hundreds or more conditions should also be familiar with concepts in analysis of high-throughput biological data. BEAN-counter encapsulates the knowledge we have accumulated from, and successfully applied to, our multiplexed, pooled barcode sequencing experiments. This protocol will be useful to those interested in generating their own high-dimensional, quantitative characterizations of gene or chemical function in a high-throughput manner.

Automated precision alignment of optical components for hydroxide catalysis bonding.

We describe an interferometric system that can measure the alignment and separation of a polished face of a optical component and an adjacent polished surface. Accuracies achieved are ∼ 1µrad for the relative angles in two orthogonal directions and ∼ 30µm in separation. We describe the use of this readout system to automate the process of hydroxide catalysis bonding of a fused-silica component to a fused-silica baseplate. The complete alignment and bonding sequence was typically achieved in a timescale of a few minutes, followed by an initial cure of 10 minutes. A series of bonds were performed using two fluids - a simple sodium hydroxide solution and a sodium hydroxide solution with some sodium silicate solution added. In each case we achieved final bonded component angular alignment within 10 µrad and position in the critical direction within 4 µm of the planned targets. The small movements of the component during the initial bonding and curing phases were monitored. The bonds made using the sodium silicate mixture achieved their final bonded alignment over a period of ∼ 15 hours. Bonds using the simple sodium hydroxide solution achieved their final alignment in a much shorter time of a few minutes. The automated system promises to speed the manufacture of precision-aligned assemblies using hydroxide catalysis bonding by more than an order of magnitude over the more manual approach used to build the optical interferometer at the heart of the recent ESA LISA Pathfinder technology demonstrator mission. This novel approach will be key to the time-efficient and low-risk manufacture of the complex optical systems needed for the forthcoming ESA spaceborne gravitational waves observatory mission, provisionally named LISA.

Integrating genetic and protein-protein interaction networks maps a functional wiring diagram of a cell.

Systematic experimental approaches have led to construction of comprehensive genetic and protein-protein interaction networks for the budding yeast, Saccharomyces cerevisiae. Genetic interactions capture functional relationships between genes using phenotypic readouts, while protein-protein interactions identify physical connections between gene products. These complementary, and largely non-overlapping, networks provide a global view of the functional architecture of a cell, revealing general organizing principles, many of which appear to be evolutionarily conserved. Here, we focus on insights derived from the integration of large-scale genetic and protein-protein interaction networks, highlighting principles that apply to both unicellular and more complex systems, including human cells. Network integration reveals fundamental connections involving key functional modules of eukaryotic cells, defining a core network of cellular function, which could be elaborated to explore cell-type specificity in metazoans.

Optical fiber couplers for precision spaceborne metrology.

We describe the optical and mechanical design, construction philosophy, and testing of a pair of matched, spaceflight-qualified fiber couplers. The couplers were developed for the LISA Pathfinder mission but are relevant for other applications-both on ground and in space-where a robust fiber coupler with well-controlled beam parameters and stable beam pointing is required. This particular implementation of the design called for two couplers providing collimated beams with individual waist sizes and positions. The target values were a 522 µm waist 145 mm after the collimating lens for one coupler and a virtual 520 µm waist 194 mm before the collimating lens for the second coupler. Values of (542±4) µm at (142±19) mm and (500±8) µm at (-275±8) mm were achieved, fully meeting the mission requirements. To control spurious noise effects in the interferometer, the optical system design also specified tight limits on relative beam curvature at an intended interference point. With nominal curvatures at this location of ∼2.35 m, the matching between the outputs of the two fiber couplers was measured to be λ/33 peak-valley over the central 1 mm of the beams. Results showing pointing stability of 3 µrad/°C over a 50°C range are presented. The vibration, shock, and thermal vacuum environmental testing conditions to which a pair of qualification fiber couplers were subjected-without change in performance-are listed.

NINJA-OPS: Fast Accurate Marker Gene Alignment Using Concatenated Ribosomes.

The explosion of bioinformatics technologies in the form of next generation sequencing (NGS) has facilitated a massive influx of genomics data in the form of short reads. Short read mapping is therefore a fundamental component of next generation sequencing pipelines which routinely match these short reads against reference genomes for contig assembly. However, such techniques have seldom been applied to microbial marker gene sequencing studies, which have mostly relied on novel heuristic approaches. We propose NINJA Is Not Just Another OTU-Picking Solution (NINJA-OPS, or NINJA for short), a fast and highly accurate novel method enabling reference-based marker gene matching (picking Operational Taxonomic Units, or OTUs). NINJA takes advantage of the Burrows-Wheeler (BW) alignment using an artificial reference chromosome composed of concatenated reference sequences, the "concatesome," as the BW input. Other features include automatic support for paired-end reads with arbitrary insert sizes. NINJA is also free and open source and implements several pre-filtering methods that elicit substantial speedup when coupled with existing tools. We applied NINJA to several published microbiome studies, obtaining accuracy similar to or better than previous reference-based OTU-picking methods while achieving an order of magnitude or more speedup and using a fraction of the memory footprint. NINJA is a complete pipeline that takes a FASTA-formatted input file and outputs a QIIME-formatted taxonomy-annotated BIOM file for an entire MiSeq run of human gut microbiome 16S genes in under 10 minutes on a dual-core laptop.

Precision absolute positional measurement of laser beams.

We describe an instrument which, coupled with a suitable coordinate measuring machine, facilitates the absolute measurement within the machine frame of the propagation direction of a millimeter-scale laser beam to an accuracy of around ±4 µm in position and ±20 µrad in angle.

Construction of rugged, ultrastable optical assemblies with optical component alignment at the few microradian level.

A method for constructing quasimonolithic, precision-aligned optical assemblies is presented. Hydroxide-catalysis bonding is used, adapted to allow optimization of component fine alignment prior to the bond setting. We demonstrate the technique by bonding a fused silica mirror substrate to a fused silica baseplate. In-plane component placement at the submicrometer level is achieved, resulting in angular control of a reflected laser beam at the sub-10-µrad level. Within the context of the LISA Pathfinder mission, the technique has been demonstrated as suitable for use in space-flight applications. It is expected that there will also be applications in a wide range of areas where accuracy, stability, and strength of optical assemblies are important.