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1.
Elife ; 122023 08 08.
Article in English | MEDLINE | ID: mdl-37552050

ABSTRACT

Loss-of-function genetic tools are widely applied for validating therapeutic targets, but their utility remains limited by incomplete on- and uncontrolled off-target effects. We describe artificial RNA interference (ARTi) based on synthetic, ultra-potent, off-target-free shRNAs that enable efficient and inducible suppression of any gene upon introduction of a synthetic target sequence into non-coding transcript regions. ARTi establishes a scalable loss-of-function tool with full control over on- and off-target effects.


Subject(s)
RNA Interference , RNA, Small Interfering/genetics
2.
Cell Rep ; 39(2): 110636, 2022 04 12.
Article in English | MEDLINE | ID: mdl-35417719

ABSTRACT

Genetic networks are characterized by extensive buffering. During tumor evolution, disruption of functional redundancies can create de novo vulnerabilities that are specific to cancer cells. Here, we systematically search for cancer-relevant paralog interactions using CRISPR screens and publicly available loss-of-function datasets. Our analysis reveals >2,000 candidate dependencies, several of which we validate experimentally, including CSTF2-CSTF2T, DNAJC15-DNAJC19, FAM50A-FAM50B, and RPP25-RPP25L. We provide evidence that RPP25L can physically and functionally compensate for the absence of RPP25 as a member of the RNase P/MRP complexes in tRNA processing. Our analysis also reveals unexpected redundancies between sex chromosome genes. We show that chrX- and chrY-encoded paralogs, such as ZFX-ZFY, DDX3X-DDX3Y, and EIF1AX-EIF1AY, are functionally linked. Tumor cell lines from male patients with loss of chromosome Y become dependent on the chrX-encoded gene. We propose targeting of chrX-encoded paralogs as a general therapeutic strategy for human tumors that have lost the Y chromosome.


Subject(s)
Neoplasms , Oncogenes , DEAD-box RNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Male , Minor Histocompatibility Antigens/metabolism , Neoplasms/genetics , RNA-Binding Proteins/genetics , Sex Chromosomes/metabolism , X Chromosome , Y Chromosome
3.
Cell Rep ; 20(2): 411-426, 2017 07 11.
Article in English | MEDLINE | ID: mdl-28700942

ABSTRACT

Cell migration through the brain parenchyma underpins neurogenesis and glioblastoma (GBM) development. Since GBM cells and neuroblasts use the same migratory routes, mechanisms underlying migration during neurogenesis and brain cancer pathogenesis may be similar. Here, we identify a common pathway controlling cell migration in normal and neoplastic cells in the CNS. The nuclear scaffold protein promyelocytic leukemia (PML), a regulator of forebrain development, promotes neural progenitor/stem cell (NPC) and neuroblast migration in the adult mouse brain. The PML pro-migratory role is active also in transformed mouse NPCs and in human primary GBM cells. In both normal and neoplastic settings, PML controls cell migration via Polycomb repressive complex 2 (PRC2)-mediated repression of Slits, key regulators of axon guidance. Finally, a PML/SLIT1 axis regulates sensitivity to the PML-targeting drug arsenic trioxide in primary GBM cells. Taken together, these findings uncover a drug-targetable molecular axis controlling cell migration in both normal and neoplastic cells.


Subject(s)
Central Nervous System/metabolism , Promyelocytic Leukemia Protein/metabolism , Animals , Cell Differentiation/genetics , Cell Differentiation/physiology , Cell Movement/physiology , Cells, Cultured , Central Nervous System/cytology , Glioblastoma/metabolism , Histone-Lysine N-Methyltransferase/metabolism , Humans , Mice , Neurogenesis/genetics , Neurogenesis/physiology , Nuclear Lamina/metabolism
4.
Nat Commun ; 6: 10237, 2015 Dec 17.
Article in English | MEDLINE | ID: mdl-26674669

ABSTRACT

Genome engineering has been greatly enhanced by the availability of Cas9 endonuclease that can be targeted to almost any genomic locus using so called guide RNAs (gRNAs). However, the introduction of foreign DNA sequences to tag an endogenous gene is still cumbersome as it requires the synthesis or cloning of homology templates. Here we present a strategy that enables the tagging of endogenous loci using one generic donor plasmid. It contains the tag of interest flanked by two gRNA recognition sites that allow excision of the tag from the plasmid. Co-transfection of cells with Cas9, a gRNA specifying the genomic locus of interest, the donor plasmid and a cassette-specific gRNA triggers the insertion of the tag by a homology-independent mechanism. The strategy is efficient and delivers clones that display a predictable integration pattern. As showcases we generated NanoLuc luciferase- and TurboGFP-tagged reporter cell lines.


Subject(s)
CRISPR-Cas Systems/genetics , DNA/genetics , Genetic Engineering/methods , Genome, Human/genetics , RNA, Guide, Kinetoplastida/genetics , Bacterial Proteins , CRISPR-Associated Protein 9 , Cell Line , Deoxyribonuclease I , Endonucleases , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Humans , Luciferases/genetics , Microscopy, Fluorescence , Plasmids , Reverse Transcriptase Polymerase Chain Reaction
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