RESUMO
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
Genetic interactions mediate the emergence of phenotype from genotype, but technologies for combinatorial genetic perturbation in mammalian cells are challenging to scale. Here, we identify background-independent paralog synthetic lethals from previous CRISPR genetic interaction screens, and find that the Cas12a platform provides superior sensitivity and assay replicability. We develop the in4mer Cas12a platform that uses arrays of four independent guide RNAs targeting the same or different genes. We construct a genome-scale library, Inzolia, that is ~30% smaller than a typical CRISPR/Cas9 library while also targeting ~4000 paralog pairs. Screens in cancer cells demonstrate discrimination of core and context-dependent essential genes similar to that of CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes. Importantly, the in4mer platform offers a fivefold reduction in library size compared to other genetic interaction methods, substantially reducing the cost and effort required for these assays.
Assuntos
Proteínas de Bactérias , Sistemas CRISPR-Cas , Endodesoxirribonucleases , Técnicas de Inativação de Genes , Humanos , Técnicas de Inativação de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas/genética , Biblioteca Gênica , Linhagem Celular Tumoral , Genes Essenciais , Células HEK293 , Epistasia Genética , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismoRESUMO
Whole exome and genome sequencing, coupled with refined bioinformatic pipelines, have enabled improved diagnostic yields for individuals with Mendelian conditions and have led to the rapid identification of novel syndromes. For many Mendelian neurodevelopmental disorders (NDDs), there is a lack of pre-existing model systems for mechanistic work. Thus, it is critical for translational researchers to have an accessible phenotype- and genotype-informed approach for model system selection. Single-cell RNA sequencing data can be informative in such an approach, as it can indicate which cell types express a gene of interest at the highest levels across time. For Mendelian NDDs, such data for the developing human brain is especially useful. A valuable single-cell RNA sequencing dataset of the second trimester developing human brain was produced by Bhaduri et al in 2021, but access to these data can be limited by computing power and the learning curve of single-cell data analysis. To reduce these barriers for translational research on Mendelian NDDs, we have built the web-based tool, Neurodevelopment in Trimester 2 - VIsualization of Single cell Data Online Tool (NeuroTri2-VISDOT), for exploring this single-cell dataset, and we have employed it in several different settings to demonstrate its utility for the translational research community.
RESUMO
Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research.
Assuntos
Histonas , Fenótipo , Humanos , Masculino , Feminino , Histonas/genética , Criança , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Pré-Escolar , Adolescente , Adulto , Deficiência Intelectual/genética , Deficiência Intelectual/patologiaRESUMO
Genetic interactions mediate the emergence of phenotype from genotype, but initial technologies for combinatorial genetic perturbation in mammalian cells suffer from inefficiency and are challenging to scale. Recent focus on paralog synthetic lethality in cancer cells offers an opportunity to evaluate different approaches and improve on the state of the art. Here we report a meta-analysis of CRISPR genetic interactions screens, identifying a candidate set of background-independent paralog synthetic lethals, and find that the Cas12a platform provides superior sensitivity and assay replicability. We demonstrate that Cas12a can independently target up to four genes from a single guide array, and we build on this knowledge by constructing a genome-scale library that expresses arrays of four guides per clone, a platform we call 'in4mer'. Our genome-scale human library, with only 49k clones, is substantially smaller than a typical CRISPR/Cas9 monogenic library while also targeting more than four thousand paralog pairs, triples, and quads. Proof of concept screens in four cell lines demonstrate discrimination of core and context-dependent essential genes similar to that of state-of-the-art CRISPR/Cas9 libraries, as well as detection of synthetic lethal and masking/buffering genetic interactions between paralogs of various family sizes, a capability not offered by any extant library. Importantly, the in4mer platform offers a fivefold reduction in the number of clones required to assay genetic interactions, dramatically improving the cost and effort required for these studies.
RESUMO
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
CRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype.
Assuntos
Leucemia Mieloide Aguda/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/genética , Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Inibidor de Quinase Dependente de Ciclina p21/genética , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Metabolismo dos Lipídeos/genética , Metabolismo dos Lipídeos/fisiologia , Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/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.