RESUMO
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
Assuntos
Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Vetores Genéticos/genéticaRESUMO
The diversity of CRISPR systems, coupled with scientific ingenuity, has led to an explosion of applications; however, to test newly-described innovations in their model systems, researchers typically embark on cumbersome, one-off cloning projects to generate custom reagents that are optimized for their biological questions. Here, we leverage Golden Gate cloning to create the Fragmid toolkit, a modular set of CRISPR cassettes and delivery technologies, along with a web portal, resulting in a combinatorial platform that enables scalable vector assembly within days. We further demonstrate that multiple CRISPR technologies can be assessed in parallel in a pooled screening format using this resource, enabling the rapid optimization of both novel technologies and cellular models. These results establish Fragmid as a robust system for the rapid design of CRISPR vectors, and we anticipate that this assembly approach will be broadly useful for systematic development, comparison, and dissemination of CRISPR technologies.
RESUMO
Cas12a CRISPR technology, unlike Cas9, allows for facile multiplexing of guide RNAs from a single transcript, simplifying combinatorial perturbations. While Cas12a has been implemented for multiplexed knockout genetic screens, it has yet to be optimized for CRISPR activation (CRISPRa) screens in human cells. Here, we develop a new Cas12a-based transactivation domain (TAD) recruitment system using the ALFA nanobody and demonstrate simultaneous activation of up to four genes. We screen a genome-wide library to identify modulators of growth and MEK inhibition, and we compare these results with those obtained with open reading frame (ORF) overexpression and Cas9-based CRISPRa. We find that the activity of multiplexed arrays is largely predictable from the best-performing guide and provide criteria for selecting active guides. We anticipate that these results will greatly accelerate the exploration of gene function and combinatorial phenotypes at scale.
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
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
A nursing student shares her adolescent experience of Hodgkin's lymphoma and calling from God into nursing.
