Protocol for iterative enrichment of integrated sgRNAs via derivative CRISPR-Cas9 libraries from genomic DNA of sorted fixed cells.

Here, we present a protocol for iterative enrichment of integrated single guide RNA (sgRNA) via derivative CRISPR-Cas9 from genomic DNA (gDNA) of phenotypically sorted fixed cells. We describe steps for high-scale lentiviral production, genome-wide screening, extracting gDNA from fixed cells, cloning of integrated sgRNAs, and high-scale transformation. This protocol introduces three key advantages: (1) applicability to fixed cells, (2) bypassing epigenetic drift, and (3) pause points lowering the contamination risk.

A genome-wide CRISPR/Cas9 screen identifies calreticulin as a selective repressor of ATF6α.

Activating transcription factor 6 (ATF6) is one of three endoplasmic reticulum (ER) transmembrane stress sensors that mediate the unfolded protein response (UPR). Despite its crucial role in long-term ER stress adaptation, regulation of ATF6 alpha (α) signalling remains poorly understood, possibly because its activation involves ER-to-Golgi and nuclear trafficking. Here, we generated an ATF6α/Inositol-requiring kinase 1 (IRE1) dual UPR reporter CHO-K1 cell line and performed an unbiased genome-wide CRISPR/Cas9 mutagenesis screen to systematically profile genetic factors that specifically contribute to ATF6α signalling in the presence and absence of ER stress.

Substrate recruitment via eIF2γ enhances catalytic efficiency of a holophosphatase that terminates the integrated stress response.

Dephosphorylation of pSer51 of the α subunit of translation initiation factor 2 (eIF2α) terminates signaling in the integrated stress response (ISR). A trimeric mammalian holophosphatase comprised of a protein phosphatase 1 (PP1) catalytic subunit, the conserved C-terminally located ~70 amino acid core of a substrate-specific regulatory subunit (PPP1R15A/GADD34 or PPP1R15B/CReP) and G-actin (an essential cofactor) efficiently dephosphorylate eIF2α in vitro. Unlike their viral or invertebrate counterparts, with whom they share the conserved 70 residue core, the mammalian PPP1R15s are large proteins of more than 600 residues.

The IRE1β-mediated unfolded protein response is repressed by the chaperone AGR2 in mucin producing cells.

Effector mechanisms of the unfolded protein response (UPR) in the endoplasmic reticulum (ER) are well-characterised, but how ER proteostasis is sensed is less well understood. Here, we exploited the beta isoform of the UPR transducer IRE1, that is specific to mucin-producing cells in order to gauge the relative regulatory roles of activating ligands and repressing chaperones of the specialised ER of goblet cells. Replacement of the stress-sensing luminal domain of endogenous IRE1α in CHO cells (normally expressing neither mucin nor IRE1β) with the luminal domain of IRE1β deregulated basal IRE1 activity.

Infancy-onset diabetes caused by de-regulated AMPylation of the human endoplasmic reticulum chaperone BiP.

Dysfunction of the endoplasmic reticulum (ER) in insulin-producing beta cells results in cell loss and diabetes mellitus. Here we report on five individuals from three different consanguineous families with infancy-onset diabetes mellitus and severe neurodevelopmental delay caused by a homozygous p.(Arg371Ser) mutation in FICD.

Higher-order phosphatase-substrate contacts terminate the integrated stress response.

Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained.

Cargo receptor-assisted endoplasmic reticulum export of pathogenic α1-antitrypsin polymers.

Circulating polymers of α1-antitrypsin (α1AT) are neutrophil chemo-attractants and contribute to inflammation, yet cellular factors affecting their secretion remain obscure. We report on a genome-wide CRISPR-Cas9 screen for genes affecting trafficking of polymerogenic α1AT. A CRISPR enrichment approach based on recovery of single guide RNA (sgRNA) sequences from phenotypically selected fixed cells reveals that cells with high-polymer content are enriched in sgRNAs targeting genes involved in "cargo loading into COPII-coated vesicles," where "COPII" is coat protein II, including the cargo receptors lectin mannose binding1 (LMAN1) and surfeit protein locus 4 (SURF4).

ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B.

The small molecule ISRIB antagonizes the activation of the integrated stress response (ISR) by phosphorylated translation initiation factor 2, eIF2(αP). ISRIB and eIF2(αP) bind distinct sites in their common target, eIF2B, a guanine nucleotide exchange factor for eIF2. We have found that ISRIB-mediated acceleration of eIF2B's nucleotide exchange activity in vitro is observed preferentially in the presence of eIF2(αP) and is attenuated by mutations that desensitize eIF2B to the inhibitory effect of eIF2(αP).

Publisher Correction: GDF15 mediates the effects of metformin on body weight and energy balance.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

GDF15 mediates the effects of metformin on body weight and energy balance.

Metformin, the world's most prescribed anti-diabetic drug, is also effective in preventing type 2 diabetes in people at high risk. More than 60% of this effect is attributable to the ability of metformin to lower body weight in a sustained manner. The molecular mechanisms by which metformin lowers body weight are unknown.

The ribosomal P-stalk couples amino acid starvation to GCN2 activation in mammalian cells.

The eukaryotic translation initiation factor 2α (eIF2α) kinase GCN2 is activated by amino acid starvation to elicit a rectifying physiological program known as the Integrated Stress Response (ISR). A role for uncharged tRNAs as activating ligands of yeast GCN2 is supported experimentally. However, mouse GCN2 activation has recently been observed in circumstances associated with ribosome stalling with no global increase in uncharged tRNAs.

GDF15 Provides an Endocrine Signal of Nutritional Stress in Mice and Humans.

GDF15 is an established biomarker of cellular stress. The fact that it signals via a specific hindbrain receptor, GFRAL, and that mice lacking GDF15 manifest diet-induced obesity suggest that GDF15 may play a physiological role in energy balance. We performed experiments in humans, mice, and cells to determine if and how nutritional perturbations modify GDF15 expression.

Predicting the mutations generated by repair of Cas9-induced double-strand breaks.

The DNA mutation produced by cellular repair of a CRISPR-Cas9-generated double-strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, but depend on DNA sequence at the targeted location. Here we systematically study the influence of flanking DNA sequence on repair outcome by measuring the edits generated by >40,000 guide RNAs (gRNAs) in synthetic constructs.

Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury.

A J-Protein Co-chaperone Recruits BiP to Monomerize IRE1 and Repress the Unfolded Protein Response.

When unfolded proteins accumulate in the endoplasmic reticulum (ER), the unfolded protein response (UPR) increases ER-protein-folding capacity to restore protein-folding homeostasis. Unfolded proteins activate UPR signaling across the ER membrane to the nucleus by promoting oligomerization of IRE1, a conserved transmembrane ER stress receptor. However, the coupling of ER stress to IRE1 oligomerization and activation has remained obscure.

Defective ATG16L1-mediated removal of IRE1α drives Crohn's disease-like ileitis.

ATG16L1, a major risk polymorphism in Crohn's disease (CD), causes impaired autophagy, but it has remained unclear how this predisposes to CD. In this study, we report that mice with Atg16l1 deletion in intestinal epithelial cells (IECs) spontaneously develop transmural ileitis phenocopying ileal CD in an age-dependent manner, driven by the endoplasmic reticulum (ER) stress sensor IRE1α. IRE1α accumulates in Paneth cells of Atg16l1 mice, and humans homozygous for ATG16L1 exhibit a corresponding increase of IRE1α in intestinal epithelial crypts.

Paradoxical Sensitivity to an Integrated Stress Response Blocking Mutation in Vanishing White Matter Cells.

The eukaryotic translation initiation factor eIF2B promotes mRNA translation as a guanine nucleotide exchange factor (GEF) for translation initiation factor 2 (eIF2). Endoplasmic reticulum (ER) stress-mediated activation of the kinase PERK and the resultant phosphorylation of eIF2's alpha subunit (eIF2α) attenuates eIF2B GEF activity thereby inducing an integrated stress response (ISR) that defends against protein misfolding in the ER. Mutations in all five subunits of human eIF2B cause an inherited leukoencephalopathy with vanishing white matter (VWM), but the role of the ISR in its pathogenesis remains unclear.

Dual role of the integrated stress response in medulloblastoma tumorigenesis.

In response to endoplasmic reticulum (ER) stress, activation of pancreatic ER kinase (PERK) coordinates an adaptive program known as the integrated stress response (ISR) by phosphorylating translation initiation factor 2α (eIF2α). Phosphorylated eIF2α is quickly dephosphorylated by the protein phosphatase 1 and growth arrest and DNA damage 34 (GADD34) complex. Data indicate that the ISR can either promote or suppress tumor development.

PERK Activation Promotes Medulloblastoma Tumorigenesis by Attenuating Premalignant Granule Cell Precursor Apoptosis.

Evidence suggests that activation of pancreatic endoplasmic reticulum kinase (PERK) signaling in response to endoplasmic reticulum stress negatively or positively influences cell transformation by regulating apoptosis. Patched1 heterozygous deficient (Ptch1(+/-)) mice reproduce human Gorlin's syndrome and are regarded as the best animal model to study tumorigenesis of the sonic hedgehog subgroup of medulloblastomas. It is believed that medulloblastomas in Ptch1(+/-) mice results from the transformation of granule cell precursors (GCPs) in the developing cerebellum.

Skeletal muscle-specific eukaryotic translation initiation factor 2α phosphorylation controls amino acid metabolism and fibroblast growth factor 21-mediated non-cell-autonomous energy metabolism.

The eukaryotic translation initiation factor 2α (eIF2α) phosphorylation-dependent integrated stress response (ISR), a component of the unfolded protein response, has long been known to regulate intermediary metabolism, but the details are poorly worked out. We report that profiling of mRNAs of transgenic mice harboring a ligand-activated skeletal muscle-specific derivative of the eIF2α protein kinase R-like ER kinase revealed the expected up-regulation of genes involved in amino acid biosynthesis and transport but also uncovered the induced expression and secretion of a myokine, fibroblast growth factor 21 (FGF21), that stimulates energy consumption and prevents obesity. The link between the ISR and FGF21 expression was further reinforced by the identification of a small-molecule ISR activator that promoted Fgf21 expression in cell-based screens and by implication of the ISR-inducible activating transcription factor 4 in the process.

Physiological modulation of BiP activity by trans-protomer engagement of the interdomain linker.

DnaK/Hsp70 chaperones form oligomers of poorly understood structure and functional significance. Site-specific proteolysis and crosslinking were used to probe the architecture of oligomers formed by the endoplasmic reticulum (ER) Hsp70, BiP. These were found to consist of adjacent protomers engaging the interdomain linker of one molecule in the substrate binding site of another, attenuating the chaperone function of oligomeric BiP.

A Missense Mutation in PPP1R15B Causes a Syndrome Including Diabetes, Short Stature, and Microcephaly.

Dysregulated endoplasmic reticulum stress and phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) are associated with pancreatic β-cell failure and diabetes. Here, we report the first homozygous mutation in the PPP1R15B gene (also known as constitutive repressor of eIF2α phosphorylation [CReP]) encoding the regulatory subunit of an eIF2α-specific phosphatase in two siblings affected by a novel syndrome of diabetes of youth with short stature, intellectual disability, and microcephaly. The R658C mutation in PPP1R15B affects a conserved amino acid within the domain important for protein phosphatase 1 (PP1) binding.

Stress responses. Mutations in a translation initiation factor identify the target of a memory-enhancing compound.

The integrated stress response (ISR) modulates messenger RNA translation to regulate the mammalian unfolded protein response (UPR), immunity, and memory formation. A chemical ISR inhibitor, ISRIB, enhances cognitive function and modulates the UPR in vivo. To explore mechanisms involved in ISRIB action, we screened cultured mammalian cells for somatic mutations that reversed its effect on the ISR.

G-actin provides substrate-specificity to eukaryotic initiation factor 2α holophosphatases.

Dephosphorylation of eukaryotic translation initiation factor 2a (eIF2a) restores protein synthesis at the waning of stress responses and requires a PP1 catalytic subunit and a regulatory subunit, PPP1R15A/GADD34 or PPP1R15B/CReP. Surprisingly, PPP1R15-PP1 binary complexes reconstituted in vitro lacked substrate selectivity. However, selectivity was restored by crude cell lysate or purified G-actin, which joined PPP1R15-PP1 to form a stable ternary complex.