RESUMO
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
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
BACKGROUND: Cleft lip with or without cleft palate (CL/P) is one of the most common congenital anomalies and the etiology of orofacial clefts is multifactorial. Transforming growth factor alpha (TGFA) is expressed at the medial edge epithelium of fusing palatal shelves during craniofacial development. In this study, the association of two important TGFA gene polymorphisms, BamHI (rs11466297) and RsaI (rs3732248), with CL/P was evaluated in an Iranian population. METHODS: The frequencies of BamHI and RsaI variations were determined in 105 unrelated Iranian subjects with nonsyndromic CL/P and 218 control subjects using PCR and RFLP methods, and the results were compared with healthy controls. A p-value of <0.05 was considered statistically significant. RESULTS: The BamHI AC genotype was significantly higher (p=0.016) in the patients (12.4%) than the control group (5.0%). The BamHI C allele was significantly higher (p=0.001; OR=3.4, 95% CI: 1.6-7.4) in the cases (8.0%) compared with the control group (2.5%). CONCLUSION: Our study showed that there was an association between the TGFA BamHI variation and nonsyndromic CL/P in Iranian population.