Genome-scale exon perturbation screens uncover exons critical for cell fitness.

CRISPR-Cas technology has transformed functional genomics, yet understanding of how individual exons differentially shape cellular phenotypes remains limited. Here, we optimized and conducted massively parallel exon deletion and splice-site mutation screens in human cell lines to identify exons that regulate cellular fitness. Fitness-promoting exons are prevalent in essential and highly expressed genes and commonly overlap with protein domains and interaction interfaces.

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.

Chemical genomics with pyrvinium identifies C1orf115 as a regulator of drug efflux.

Pyrvinium is a quinoline-derived cyanine dye and an approved anti-helminthic drug reported to inhibit WNT signaling and have anti-proliferative effects in various cancer cell lines. To further understand the mechanism by which pyrvinium is cytotoxic, we conducted a pooled genome-wide CRISPR loss-of-function screen in the human HAP1 cell model. The top drug-gene sensitizer interactions implicated the malate-aspartate and glycerol-3-phosphate shuttles as mediators of cytotoxicity to mitochondrial complex I inhibition including pyrvinium.

A Method to Map Gene Essentiality of Human Pluripotent Stem Cells by Genome-Scale CRISPR Screens with Inducible Cas9.

Human pluripotent stem cells (hPSCs) have the capacity for self-renewal and differentiation into most cell types and, in contrast to widely used cell lines, are karyotypically normal and non-transformed. Hence, hPSCs are considered the gold-standard system for modelling diseases, especially in the field of regenerative medicine. Despite widespread research use of hPSCs and induced pluripotent stem cells (iPSCs), the systematic understanding of pluripotency and lineage differentiation mechanisms are still incomplete.

Application of CHyMErA Cas9-Cas12a combinatorial genome-editing platform for genetic interaction mapping and gene fragment deletion screening.

CRISPR-based forward genetic screening represents a powerful approach for the systematic characterization of gene function. Recent efforts have been directed toward establishing CRISPR-based tools for the programmable delivery of combinatorial genetic perturbations, most of which are mediated by a single nuclease and the expression of structurally identical guide backbones from two promoters. In contrast, we have developed CHyMErA (Cas hybrid for multiplexed editing and screening applications), which is based on the co-expression of Cas9 and Cas12a nucleases in conjunction with a hybrid guide RNA (hgRNA) engineered by the fusion of Cas9 and Cas12a guides and expressed from a single U6 promoter.

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.

LRRC8A-containing chloride channel is crucial for cell volume recovery and survival under hypertonic conditions.

Regulation of cell volume is essential for tissue homeostasis and cell viability. In response to hypertonic stress, cells need rapid electrolyte influx to compensate water loss and to prevent cell death in a process known as regulatory volume increase (RVI). However, the molecular component able to trigger such a process was unknown to date.

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.

Functional genomic landscape of cancer-intrinsic evasion of killing by T cells.

The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity.

Systematic mapping of genetic interactions for de novo fatty acid synthesis identifies C12orf49 as a regulator of lipid metabolism.

The de novo synthesis of fatty acids has emerged as a therapeutic target for various diseases, including cancer. Because cancer cells are intrinsically buffered to combat metabolic stress, it is important to understand how cells may adapt to the loss of de novo fatty acid biosynthesis. Here, we use pooled genome-wide CRISPR screens to systematically map genetic interactions (GIs) in human HAP1 cells carrying a loss-of-function mutation in fatty acid synthase (FASN), whose product catalyses the formation of long-chain fatty acids.

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.

Genome-wide CRISPR-Cas9 Interrogation of Splicing Networks Reveals a Mechanism for Recognition of Autism-Misregulated Neuronal Microexons.

Alternative splicing is crucial for diverse cellular, developmental, and pathological processes. However, the full networks of factors that control individual splicing events are not known. Here, we describe a CRISPR-based strategy for the genome-wide elucidation of pathways that control splicing and apply it to microexons with important functions in nervous system development and that are commonly misregulated in autism.

Pooled Lentiviral CRISPR-Cas9 Screens for Functional Genomics in Mammalian Cells.

CRISPR-Cas9 technology provides a simple way to introduce targeted mutations into mammalian cells to induce loss-of-function phenotypes. The CRISPR-Cas9 system has now successfully been applied for genetic screens in many cell types, providing a powerful tool for functional genomics with manifold applications. Genome-wide guide-RNA (gRNA) libraries allow facile generation of a pool of cells, each harboring a gene knockout mutation that can be used for the study of gene function, pathway analysis or the identification of genes required for cellular fitness.

CRISPR screens identify genomic ribonucleotides as a source of PARP-trapping lesions.

The observation that BRCA1- and BRCA2-deficient cells are sensitive to inhibitors of poly(ADP-ribose) polymerase (PARP) has spurred the development of cancer therapies that use these inhibitors to target deficiencies in homologous recombination. The cytotoxicity of PARP inhibitors depends on PARP trapping, the formation of non-covalent protein-DNA adducts composed of inhibited PARP1 bound to DNA lesions of unclear origins. To address the nature of such lesions and the cellular consequences of PARP trapping, we undertook three CRISPR (clustered regularly interspersed palindromic repeats) screens to identify genes and pathways that mediate cellular resistance to olaparib, a clinically approved PARP inhibitor.

Evaluation and Design of Genome-Wide CRISPR/SpCas9 Knockout Screens.

The adaptation of CRISPR/SpCas9 technology to mammalian cell lines is transforming the study of human functional genomics. Pooled libraries of CRISPR guide RNAs (gRNAs) targeting human protein-coding genes and encoded in viral vectors have been used to systematically create gene knockouts in a variety of human cancer and immortalized cell lines, in an effort to identify whether these knockouts cause cellular fitness defects. Previous work has shown that CRISPR screens are more sensitive and specific than pooled-library shRNA screens in similar assays, but currently there exists significant variability across CRISPR library designs and experimental protocols.

Regulation of mRNA capping in the cell cycle.

The mRNA cap structure, which is added to nascent RNA pol II transcripts, recruits the protein complexes required for pre-mRNA transcript processing, mRNA export and translation initiation. The enzymes which catalyze mRNA cap synthesis are regulated by cellular signaling pathways which impact on their expression, localization and activity. Here we discuss the recent observation that the mRNA cap methyltransferase, RNMT, is phosphorylated on Thr-77 by CDK1-cyclin B1, which regulates its activity and the proteins with which it interacts.

CDK1-Cyclin B1 Activates RNMT, Coordinating mRNA Cap Methylation with G1 Phase Transcription.

The creation of translation-competent mRNA is dependent on RNA polymerase II transcripts being modified by addition of the 7-methylguanosine (m7G) cap. The factors that mediate splicing, nuclear export, and translation initiation are recruited to the transcript via the cap. The cap structure is formed by several activities and completed by RNMT (RNA guanine-7 methyltransferase), which catalyzes N7 methylation of the cap guanosine.

High-Resolution CRISPR Screens Reveal Fitness Genes and Genotype-Specific Cancer Liabilities.

The ability to perturb genes in human cells is crucial for elucidating gene function and holds great potential for finding therapeutic targets for diseases such as cancer. To extend the catalog of human core and context-dependent fitness genes, we have developed a high-complexity second-generation genome-scale CRISPR-Cas9 gRNA library and applied it to fitness screens in five human cell lines. Using an improved Bayesian analytical approach, we consistently discover 5-fold more fitness genes than were previously observed.

Extensive mapping of an innate immune network with CRISPR.

The application of the CRISPR‐Cas9 system marks a major breakthrough for genetic screens, particularly in mammalian cells where high‐throughput targeted gene editing has been lacking. Parnas et al (2015) apply this screening technology to mouse bone marrow‐derived dendritic cells in order to study the regulation of the immune response triggered by PAMPs. Through integrated analysis of gene knockouts in conjunction with changes in protein and mRNA expression, CRISPR screens are facilitating dissection of immune regulatory networks at unprecedented resolution.

Human cap methyltransferase (RNMT) N-terminal non-catalytic domain mediates recruitment to transcription initiation sites.

Gene expression in eukaryotes is dependent on the mRNA methyl cap which mediates mRNA processing and translation initiation. Synthesis of the methyl cap initiates with the addition of 7-methylguanosine to the initiating nucleotide of RNA pol II (polymerase II) transcripts, which occurs predominantly during transcription and in mammals is catalysed by RNGTT (RNA guanylyltransferase and 5' phosphatase) and RNMT (RNA guanine-7 methyltransferase). RNMT has a methyltransferase domain and an N-terminal domain whose function is unclear; it is conserved in mammals, but not required for cap methyltransferase activity.

Primary and secondary siRNAs in geminivirus-induced gene silencing.

In plants, RNA silencing-based antiviral defense is mediated by Dicer-like (DCL) proteins producing short interfering (si)RNAs. In Arabidopsis infected with the bipartite circular DNA geminivirus Cabbage leaf curl virus (CaLCuV), four distinct DCLs produce 21, 22 and 24 nt viral siRNAs. Using deep sequencing and blot hybridization, we found that viral siRNAs of each size-class densely cover the entire viral genome sequences in both polarities, but highly abundant siRNAs correspond primarily to the leftward and rightward transcription units.

E2F1-dependent methyl cap formation requires RNA pol II phosphorylation.

Gene expression is a process integral to cell proliferation. The E2F family of transcription factors upregulates expression of transcripts whose products are essential for cell cycle progression. Here, we report that E2F1 promotes gene expression by an additional mechanism, that is, formation of the methyl cap on RNA pol II transcripts.

Sequencing of RDR6-dependent double-stranded RNAs reveals novel features of plant siRNA biogenesis.

Biogenesis of trans-acting siRNAs (tasiRNAs) is initiated by miRNA-directed cleavage of TAS gene transcripts and requires RNA-dependent RNA polymerase 6 (RDR6) and Dicer-like 4 (DCL4). Here, we show that following miR173 cleavage the entire polyadenylated parts of Arabidopsis TAS1a/b/c and TAS2 transcripts are converted by RDR6 to double-stranded (ds)RNAs. Additionally, shorter dsRNAs are produced following a second cleavage directed by a TAS1c-derived siRNA.