Unraveling the mechanism of action and molecular target of small molecules remains a major challenge in drug discovery. While many cancer drugs target genetic vulnerabilities, loss-of-function ...screens fail to identify essential genes in drug mechanism of action. Here, we report CRISPRres, a CRISPR-Cas-based genetic screening approach to rapidly derive and identify drug resistance mutations in essential genes. It exploits the local genetic variation created by CRISPR-Cas-induced non-homologous end-joining (NHEJ) repair to generate a wide variety of functional in-frame mutations. Using large sgRNA tiling libraries and known drug-target pairs, we validate it as a target identification approach. We apply CRISPRres to the anticancer agent KPT-9274 and identify nicotinamide phosphoribosyltransferase (NAMPT) as its main target. These results present a powerful and simple genetic approach to create many protein variants that, in combination with positive selection, can be applied to reveal the cellular target of small-molecule inhibitors.
A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for ...the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for beta-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbbeta and GPIX but also a significantly lower expression of the nondirectly GATA1-regulated Gsalpha gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbalpha, GPIbbeta, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion, GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
A new mutation is described in the X-linked gene GATA1, resulting in macrothrombocytopenia and mild dyserythropoietic features but no marked anemia in a 4-generation family. The molecular basis for ...the observed phenotype is a substitution of glycine for aspartate in the strictly conserved codon 218 (D218G) of the amino-terminal zinc finger loop of the transcription factor GATA1. Zinc finger interaction studies demonstrated that this mutation results in a weak loss of affinity of GATA1 for its essential cofactor FOG1, whereas direct D218G-GATA1 binding to DNA was normal. The phenotypic effects of this mutation in the patients' platelets have been studied. Semiquantitative RNA analysis, normalized for β-actin messenger RNA, showed extremely low transcription of the GATA1 target genes GPIbβ and GPIXbut also a significantly lower expression of the nondirectly GATA1-regulated Gsα gene, suggestive of incomplete megakaryocyte maturation. In contrast, GPIIIa expression was close to normal in agreement with its early appearance during megakaryocyte differentiation. Flow cytometric analysis of patient platelets confirmed the existence of a platelet population with abnormal size distribution and reduced GPIb complex levels but with normal GPIIIa expression. It also showed the presence of very immature platelets lacking almost all membrane glycoproteins studied (GPIbα, GPIbβ, GPIIIa, GPIX, and GPV). Patients' platelets showed weak ristocetin-induced agglutination, compatible with the disturbed GPIb complex. Accordingly, electron microscopy of the patients' platelets revealed giant platelets with cytoplasmic clusters consisting of smooth endoplasmic reticulum and abnormal membrane complexes. In conclusion,GATA1 mutations can lead to isolated X-linked macrothrombocytopenia without anemia.
PDF
