RESUMO
Chromatin regulators are frequently mutated in human cancer and are attractive drug targets. They include diverse proteins that share functional domains and assemble into related multi-subunit complexes. To investigate functional relationships among these regulators, here we apply combinatorial CRISPR knockouts (KOs) to test over 35,000 gene-gene pairings in leukemia cells, using a library of over 300,000 constructs. Top pairs that demonstrate either compensatory non-lethal interactions or synergistic lethality enrich for paralogs and targets that occupy the same protein complex. The screen highlights protein complex dependencies not apparent in single KO screens, for example MCM histone exchange, the nucleosome remodeling and deacetylase (NuRD) complex, and HBO1 (KAT7) complex. We explore two approaches to NuRD complex inactivation. Paralog and non-paralog combinations of the KAT7 complex emerge as synergistic lethal and specifically nominate the ING5 PHD domain as a potential therapeutic target when paired with other KAT7 complex member losses. These findings highlight the power of combinatorial screening to provide mechanistic insight and identify therapeutic targets within redundant networks.
Assuntos
Cromatina , Leucemia , Humanos , Cromatina/genética , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Montagem e Desmontagem da Cromatina , Leucemia/tratamento farmacológico , Leucemia/genética , Histona Acetiltransferases/metabolismoRESUMO
Numerous rationally-designed and directed-evolution variants of SpCas9 have been reported to expand the utility of CRISPR technology. Here, we assess the activity and specificity of WT-Cas9 and 10 SpCas9 variants by benchmarking their PAM preferences, on-target activity, and off-target susceptibility in cell culture assays with thousands of guides targeting endogenous genes. To enhance the coverage and thus utility of base editing screens, we demonstrate that the SpCas9-NG and SpG variants are compatible with both A > G and C > T base editors, more than tripling the number of guides and assayable residues. We demonstrate the performance of these technologies by screening for loss-of-function mutations in BRCA1 and Venetoclax-resistant mutations in BCL2, identifying both known and new mutations that alter function. We anticipate that the tools and methodologies described here will facilitate the investigation of genetic variants at a finer and deeper resolution for any locus of interest.
Assuntos
Proteína 9 Associada à CRISPR , Edição de Genes , Benchmarking , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Understanding the functional consequences of single-nucleotide variants is critical to uncovering the genetic underpinnings of diseases, but technologies to characterize variants are limiting. Here, we leverage CRISPR-Cas9 cytosine base editors in pooled screens to scalably assay variants at endogenous loci in mammalian cells. We benchmark the performance of base editors in positive and negative selection screens, identifying known loss-of-function mutations in BRCA1 and BRCA2 with high precision. To demonstrate the utility of base editor screens to probe small molecule-protein interactions, we screen against BH3 mimetics and PARP inhibitors, identifying point mutations that confer drug sensitivity or resistance. We also create a library of single guide RNAs (sgRNAs) predicted to generate 52,034 ClinVar variants in 3,584 genes and conduct screens in the presence of cellular stressors, identifying loss-of-function variants in numerous DNA damage repair genes. We anticipate that this screening approach will be broadly useful to readily and scalably functionalize genetic variants.
Assuntos
Edição de Genes , Variação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Alelos , Proteína BRCA1/genética , Proteína BRCA2/genética , Sequência de Bases , Domínio Catalítico , Linhagem Celular Tumoral , Humanos , Mutação com Perda de Função , Mutagênese/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Mutação Puntual/genética , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Reprodutibilidade dos Testes , Seleção Genética , Proteína bcl-X/genéticaRESUMO
Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript, and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. In the present study, we describe the optimization of enhanced Cas12a from Acidaminococcus (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2. Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.
Assuntos
Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas/genética , Endodesoxirribonucleases , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos , Acidaminococcus/genética , Apoptose/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteína 9 Associada à CRISPR , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Linhagem Celular Tumoral , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Biblioteca Gênica , Células HEK293 , Humanos , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is labor-intensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and small-molecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.
Assuntos
Testes Genéticos/métodos , Apoptose/genética , Sistemas CRISPR-Cas , Linhagem Celular , Células Clonais , Técnicas de Inativação de Genes , Biblioteca Gênica , Redes Reguladoras de Genes , Humanos , Família Multigênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Proteína de Sequência 1 de Leucemia de Células Mieloides/deficiência , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/deficiência , Poli(ADP-Ribose) Polimerase-1/genética , Análise de Célula Única , Proteína bcl-X/antagonistas & inibidores , Proteína bcl-X/deficiência , Proteína bcl-X/genéticaRESUMO
Unbiased in vivo genome-wide genetic screening is a powerful approach to elucidate new molecular mechanisms, but such screening has not been possible to perform in the mammalian central nervous system (CNS). Here, we report the results of the first genome-wide genetic screens in the CNS using both short hairpin RNA (shRNA) and CRISPR libraries. Our screens identify many classes of CNS neuronal essential genes and demonstrate that CNS neurons are particularly sensitive not only to perturbations to synaptic processes but also autophagy, proteostasis, mRNA processing, and mitochondrial function. These results reveal a molecular logic for the common implication of these pathways across multiple neurodegenerative diseases. To further identify disease-relevant genetic modifiers, we applied our screening approach to two mouse models of Huntington's disease (HD). Top mutant huntingtin toxicity modifier genes included several Nme genes and several genes involved in methylation-dependent chromatin silencing and dopamine signaling, results that reveal new HD therapeutic target pathways.
Assuntos
Sobrevivência Celular/genética , Proteína Huntingtina/genética , Doença de Huntington/genética , Neostriado/metabolismo , Neurônios/metabolismo , Animais , Comportamento Animal , Sistemas CRISPR-Cas , Técnicas de Silenciamento de Genes , Biblioteca Gênica , Genes Essenciais/genética , Camundongos , Camundongos Transgênicos , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo Difosfato Quinase D/genética , Agregados Proteicos , Interferência de RNA , RNA Guia de Cinetoplastídeos , RNA Interferente Pequeno , Receptores de Dopamina D2/genética , Análise de Sequência de RNARESUMO
Many implementations of pooled screens in mammalian cells rely on linking an element of interest to a barcode, with the latter subsequently quantitated by next generation sequencing. However, substantial uncoupling between these paired elements during lentiviral production has been reported, especially as the distance between elements increases. We detail that PCR amplification is another major source of uncoupling, and becomes more pronounced with increased amounts of DNA template molecules and PCR cycles. To lessen uncoupling in systems that use paired elements for detection, we recommend minimizing the distance between elements, using low and equal template DNA inputs for plasmid and genomic DNA during PCR, and minimizing the number of PCR cycles. We also present a vector design for conducting combinatorial CRISPR screens that enables accurate barcode-based detection with a single short sequencing read and minimal uncoupling.
Assuntos
Sistemas CRISPR-Cas , Código de Barras de DNA Taxonômico/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , RNA Guia de Cinetoplastídeos/genética , Linhagem Celular Tumoral , Vetores Genéticos , Humanos , Lentivirus/genética , Recombinação GenéticaRESUMO
Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
Assuntos
Sistemas CRISPR-Cas/genética , Epistasia Genética/genética , Testes Genéticos , RNA Guia de Cinetoplastídeos/genética , Apoptose/genética , Técnicas de Inativação de Genes , Marcação de Genes , Humanos , Aprendizado de Máquina , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Transdução de Sinais/genética , Staphylococcus aureus/genética , Streptococcus pyogenes/genética , Proteína Supressora de Tumor p53/genéticaRESUMO
CRISPR/Cas9 screening has proven to be a versatile tool for genomics research. Based on unexpected results from a genome-wide screen, we developed a CRISPR/Cas9-mediated approach to mutagenesis, exploiting the allelic diversity generated by error-prone non-homologous end-joining (NHEJ) to identify novel gain-of-function and drug resistant alleles of the MAPK signaling pathway genes MEK1 and BRAF. We define the parameters of a scalable technique to easily generate cell populations containing thousands of endogenous allelic variants to map gene functions. Further, these results highlight an unexpected but important phenomenon, that Cas9-induced gain-of-function alleles are an inherent by-product of normal Cas9 loss-of-function screens and should be investigated during analysis of data from large-scale positive selection screens.
Assuntos
Sistemas CRISPR-Cas , MAP Quinase Quinase 1/genética , Mutagênese , Engenharia de Proteínas/métodos , Proteínas Proto-Oncogênicas B-raf/genética , Alelos , Linhagem Celular Tumoral , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA por Junção de Extremidades , Biblioteca Gênica , Células HEK293 , Humanos , Mutação INDEL , Indóis/farmacologia , MAP Quinase Quinase 1/química , Fenótipo , Proteínas Proto-Oncogênicas B-raf/química , RNA Guia de Cinetoplastídeos/genética , Proteínas Recombinantes de Fusão/química , Alinhamento de Sequência , Sulfonamidas/farmacologia , Transdução Genética , VemurafenibRESUMO
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.