A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and TNKS/2.

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerases TNKS and TNKS2, which bind the peroxisomal membrane protein PEX14. We propose that RNF146 and TNKS/2 regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased TNKS/2 and RNF146-dependent degradation of non-peroxisomal substrates, including the ß-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of ß-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation but also a novel role in bridging peroxisome function with Wnt/ß-catenin signaling during development.

A genome-wide screen links peroxisome regulation with Wnt signaling through RNF146 and tankyrase.

Peroxisomes are membrane-bound organelles harboring metabolic enzymes. In humans, peroxisomes are required for normal development, yet the genes regulating peroxisome function remain unclear. We performed a genome-wide CRISPRi screen to identify novel factors involved in peroxisomal homeostasis. We found that inhibition of RNF146, an E3 ligase activated by poly(ADP-ribose), reduced the import of proteins into peroxisomes. RNF146-mediated loss of peroxisome import depended on the stabilization and activity of the poly(ADP-ribose) polymerase tankyrase, which binds the peroxisomal membrane protein PEX14. We propose that RNF146 and tankyrase regulate peroxisome import efficiency by PARsylation of proteins at the peroxisome membrane. Interestingly, we found that the loss of peroxisomes increased tankyrase and RNF146-dependent degradation of non-peroxisomal substrates, including the beta-catenin destruction complex component AXIN1, which was sufficient to alter the amplitude of beta-catenin transcription. Together, these observations not only suggest previously undescribed roles for RNF146 in peroxisomal regulation, but also a novel role in bridging peroxisome function with Wnt/beta-catenin signaling during development.

Interstrand crosslinking of homologous repair template DNA enhances gene editing in human cells.

We describe a strategy to boost the efficiency of gene editing via homology-directed repair (HDR) by covalently modifying the template DNA with interstrand crosslinks. Crosslinked templates (xHDRTs) increase Cas9-mediated editing efficiencies by up to fivefold in K562, HEK293T, U2OS, iPS and primary T cells. Increased editing from xHDRTs is driven by events on the template molecule and requires ataxia telangiectasia and Rad3-related (ATR) kinase and components of the Fanconi anemia pathway.

High-level correction of the sickle mutation is amplified in vivo during erythroid differentiation.

Background: A point mutation in sickle cell disease (SCD) alters one amino acid in the ß-globin subunit of hemoglobin, with resultant anemia and multiorgan damage that typically shortens lifespan by decades. Because SCD is caused by a single mutation, and hematopoietic stem cells (HSCs) can be harvested, manipulated, and returned to an individual, it is an attractive target for gene correction. Results: An optimized Cas9 ribonucleoprotein (RNP) with an ssDNA oligonucleotide donor together generated correction of at least one ß-globin allele in more than 30% of long-term engrafting human HSCs. After adopting a high-fidelity Cas9 variant, efficient correction with minimal off-target events also was observed. In vivo erythroid differentiation markedly enriches for corrected ß-globin alleles, indicating that erythroblasts carrying one or more corrected alleles have a survival advantage. Significance: These findings indicate that the sickle mutation can be corrected in autologous HSCs with an optimized protocol suitable for clinical translation.

Identification of novel HPFH-like mutations by CRISPR base editing that elevate the expression of fetal hemoglobin.

Naturally occurring point mutations in the HBG promoter switch hemoglobin synthesis from defective adult beta-globin to fetal gamma-globin in sickle cell patients with hereditary persistence of fetal hemoglobin (HPFH) and ameliorate the clinical severity. Inspired by this natural phenomenon, we tiled the highly homologous HBG proximal promoters using adenine and cytosine base editors that avoid the generation of large deletions and identified novel regulatory regions including a cluster at the -123 region. Base editing at -123 and -124 bp of HBG promoter induced fetal hemoglobin (HbF) to a higher level than disruption of well-known BCL11A binding site in erythroblasts derived from human CD34+ hematopoietic stem and progenitor cells (HSPC). We further demonstrated in vitro that the introduction of -123T > C and -124T > C HPFH-like mutations drives gamma-globin expression by creating a de novo binding site for KLF1. Overall, our findings shed light on so far unknown regulatory elements within the HBG promoter and identified additional targets for therapeutic upregulation of fetal hemoglobin.

Controlled Cycling and Quiescence Enables Efficient HDR in Engraftment-Enriched Adult Hematopoietic Stem and Progenitor Cells.

Genome editing often takes the form of either error-prone sequence disruption by non-homologous end joining (NHEJ) or sequence replacement by homology-directed repair (HDR). Although NHEJ is generally effective, HDR is often difficult in primary cells. Here, we use a combination of immunophenotyping, next-generation sequencing, and single-cell RNA sequencing to investigate and reprogram genome editing outcomes in subpopulations of adult hematopoietic stem and progenitor cells. We find that although quiescent stem-enriched cells mostly use NHEJ, non-quiescent cells with the same immunophenotype use both NHEJ and HDR. Inducing quiescence before editing results in a loss of HDR in all cell subtypes. We develop a strategy of controlled cycling and quiescence that yields a 6-fold increase in the HDR/NHEJ ratio in quiescent stem cells ex vivo and in vivo. Our results highlight the tension between editing and cellular physiology and suggest strategies to manipulate quiescent cells for research and therapeutic genome editing.

The Histone Chaperone FACT Induces Cas9 Multi-turnover Behavior and Modifies Genome Manipulation in Human Cells.

Cas9 is a prokaryotic RNA-guided DNA endonuclease that binds substrates tightly in vitro but turns over rapidly when used to manipulate genomes in eukaryotic cells. Little is known about the factors responsible for dislodging Cas9 or how they influence genome engineering. Unbiased detection through proximity labeling of transient protein interactions in cell-free Xenopus laevis egg extract identified the dimeric histone chaperone facilitates chromatin transcription (FACT) as an interactor of substrate-bound Cas9. FACT is both necessary and sufficient to displace dCas9, and FACT immunodepletion converts Cas9's activity from multi-turnover to single turnover. In human cells, FACT depletion extends dCas9 residence times, delays genome editing, and alters the balance between indel formation and homology-directed repair. FACT knockdown also increases epigenetic marking by dCas9-based transcriptional effectors with a concomitant enhancement of transcriptional modulation. FACT thus shapes the intrinsic cellular response to Cas9-based genome manipulation most likely by determining Cas9 residence times.

Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts.

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1 kb). Splicing reporter studies have shown that decoy splice sites are essential for activity, suggesting that decoys act by engaging intron-terminal splice sites and competing with cross-intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides may be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that targeting a decoy 5' splice site in the O-GlcNAc transferase (OGT) gene reduced IR from ∼80% to ∼20% in primary human erythroblasts, accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, other retained introns were strongly dependent on decoy function, since antisense targeting of decoy 5' splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, α-spectrin (SPTA1). These results show that modulating decoy exon function can dramatically alter IR and suggest that dynamic regulation of decoy exons could be a mechanism to fine-tune gene expression post-transcriptionally in many cell types.

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq.

CRISPR-Cas genome editing induces targeted DNA damage but can also affect off-target sites. Current off-target discovery methods work using purified DNA or specific cellular models but are incapable of direct detection in vivo. We developed DISCOVER-Seq (discovery of in situ Cas off-targets and verification by sequencing), a universally applicable approach for unbiased off-target identification that leverages the recruitment of DNA repair factors in cells and organisms. Tracking the precise recruitment of MRE11 uncovers the molecular nature of Cas activity in cells with single-base resolution. DISCOVER-Seq works with multiple guide RNA formats and types of Cas enzymes, allowing characterization of new editing tools. Off-targets can be identified in cell lines and patient-derived induced pluripotent stem cells and during adenoviral editing of mice, paving the way for in situ off-target discovery within individual patient genotypes during therapeutic genome editing.

A rapid methods development workflow for high-throughput quantitative proteomic applications.

Recent improvements in the speed and sensitivity of liquid chromatography-mass spectrometry systems have driven significant progress toward system-wide characterization of the proteome of many species. These efforts create large proteomic datasets that provide insight into biological processes and identify diagnostic proteins whose abundance changes significantly under different experimental conditions. Yet, these system-wide experiments are typically the starting point for hypothesis-driven, follow-up experiments to elucidate the extent of the phenomenon or the utility of the diagnostic marker, wherein many samples must be analyzed. Transitioning from a few discovery experiments to quantitative analyses on hundreds of samples requires significant resources both to develop sensitive and specific methods as well as analyze them in a high-throughput manner. To aid these efforts, we developed a workflow using data acquired from discovery proteomic experiments, retention time prediction, and standard-flow chromatography to rapidly develop targeted proteomic assays. We demonstrated this workflow by developing MRM assays to quantify proteins of multiple metabolic pathways from multiple microbes under different experimental conditions. With this workflow, one can also target peptides in scheduled/dynamic acquisition methods from a shotgun proteomic dataset downloaded from online repositories, validate with appropriate control samples or standard peptides, and begin analyzing hundreds of samples in only a few minutes.

CRISPR-Cas9 interrogation of a putative fetal globin repressor in human erythroid cells.

Sickle Cell Disease and ß-thalassemia, which are caused by defective or deficient adult ß-globin (HBB) respectively, are the most common serious genetic blood diseases in the world. Persistent expression of the fetal ß-like globin, also known as 𝛾-globin, can ameliorate both disorders by serving in place of the adult ß-globin as a part of the fetal hemoglobin tetramer (HbF). Here we use CRISPR-Cas9 gene editing to explore a potential 𝛾-globin silencer region upstream of the δ-globin gene identified by comparison of naturally-occurring deletion mutations associated with up-regulated 𝛾-globin. We find that deletion of a 1.7 kb consensus element or select 350 bp sub-regions from bulk populations of cells increases levels of HbF. Screening of individual sgRNAs in one sub-region revealed three single guides that caused increases in 𝛾-globin expression. Deletion of the 1.7 kb region in HUDEP-2 clonal sublines, and in colonies derived from CD34+ hematopoietic stem/progenitor cells (HSPCs), does not cause significant up-regulation of 𝛾-globin. These data suggest that the 1.7 kb region is not an autonomous 𝛾-globin silencer, and thus by itself is not a suitable therapeutic target for gene editing treatment of ß-hemoglobinopathies.

Probing the Flexibility of an Iterative Modular Polyketide Synthase with Non-Native Substrates in Vitro.

In the search for molecular machinery for custom biosynthesis of valuable compounds, the modular type I polyketide synthases (PKSs) offer great potential. In this study, we investigate the flexibility of BorM5, the iterative fifth module of the borrelidin synthase, with a panel of non-native priming substrates in vitro. BorM5 differentially extends various aliphatic and substituted substrates. Depending on substrate size and substitution BorM5 can exceed the three iterations it natively performs. To probe the effect of methyl branching on chain length regulation, we engineered a BorM5 variant capable of incorporating methylmalonyl- and malonyl-CoA into its intermediates. Intermediate methylation did not affect overall chain length, indicating that the enzyme does not to count methyl branches to specify the number of iterations. In addition to providing regulatory insight about BorM5, we produced dozens of novel methylated intermediates that might be used for production of various hydrocarbons or pharmaceuticals. These findings enable rational engineering and recombination of BorM5 and inform the study of other iterative modules.

Superior mesenteric vein aneurysm: a case report.

A 46-year-old female was found to have a saccular superior mesenteric vein (SMV) aneurysm on computed tomography (CT) scan during workup for abdominal pain. It measured 3.5 cm in diameter. The SMV aneurysm was successfully resected, and the SMV was repaired with femoral vein patch angioplasty. She was placed on coumadin for 3 months. At follow-up, the vein patch repair was patent and the patient was doing well with complete resolution of her abdominal pain.

Redefining the role of arterial imaging in the management of penetrating zone 3 neck injuries.

The purpose of this study was to assess the role of arteriography (AG) in the diagnosis and treatment of vascular trauma in patients with zone 3 penetrating neck injuries. The records of all cases of penetrating neck trauma for the past 14 years at a level 1 trauma center were reviewed retrospectively. Eight hundred forty-four penetrating neck injuries were documented, of which 72 (8.5%) traversed zone 3 of the neck (gunshot, 35; stab, 32; shotgun, 5). Twenty patients (27%) had hard signs of vascular injury (hemorrhage, expanding hematoma, bruit, thrill, neurologic deficit). Twelve of these (60%) underwent immediate exploration, 1 had no significant injury, and 11 had successful surgical repair or ligation of the vascular injury. AG in the other eight patients with hard signs revealed injuries requiring embolization (three patients), urokinase infusion (one patient), and observation (three patients) and one normal examination. Fifty-two patients had no hard signs of vascular injury. Twenty-four of these underwent AG, of which 18 were negative. Positive findings included internal carotid artery narrowings (two patients), external compression of the internal carotid artery (one patient), vertebral artery intimal flap (one patient), and nonbleeding injuries to small, noncritical arteries (two patients), none of which required treatment. Twenty-four of the remaining 28 patients were observed clinically, and 4 patients had negative explorations. Nine patients had ultrasonography performed, but these examinations did not yield any useful information. The absence of hard signs reliably excludes surgically significant vascular injuries in penetrating zone 3 neck trauma, suggesting that AG is not necessary. Hard signs in stable patients should mandate AG because these vascular injuries may be amenable to endovascular therapy.

Differential effects of vascular growth factors on arterial and venous angiogenesis.

OBJECTIVE: Angiogenesis, the development of new blood vessels, has become an area of increased interest for both scientific and clinical application purposes. Proangiogenic agents, such as vascular endothelial growth factor (VEGF) and naltrexone, have been shown to effectively induce new blood vessel growth. Other growth factors, such as the endogenous opioid growth factor (OGF; [Met(5)]-enkephalin) and retinoic acid, are inhibitors of angiogenesis. The differential effects on veins and arteries, however, by any vascular growth factor, have not previously been investigated. METHODS: The chick chorioallantoic membrane (CAM) assay was used for the in vivo quantitation of angiogenesis. After 3 days of incubation, fertilized chick embryos were explanted, and a 3.2-mm methylcellulose disk containing either the known angiogenic stimulators VEGF (0.2 microg, 1.0 microg) or naltrexone (0.1 microg, 5.0 microg), or the angiogenic inhibitors OGF (1.0 microg, 5.0 microg) or retinoic acid (1.0 microg) was placed onto the CAM surface. An equal volume of distilled water served as a control. After 2 days of growth, the CAM arteries and veins were identified, and images were obtained with a digital camera. Quantitative analysis of angiogenesis was performed on a 100-mm(2) area surrounding the applied disk, and the number and length of the veins and arteries were measured. RESULTS: The angiogenic stimulators VEGF and naltrexone markedly increased both the total number and length of all blood vessels as compared with control values. The mean length of blood vessels decreased, suggesting the induction of new vessel growth. VEGF and naltrexone proportionately increased vein and arterial angiogenesis, maintaining artery/vein ratios for vessel number and length that were unchanged compared with controls. The angiogenic inhibitors, OGF and retinoic acid, notably decreased the total number and length of blood vessels in the CAM preparations. However, these compounds had a disproportionately greater inhibitory effect on arterial angiogenesis as reflected in decreased artery/vein ratios for vessel number and length. CONCLUSIONS: The angiogenic stimulators VEGF and naltrexone induce development of veins and arteries in a proportional manner. In contrast, the angiogenic inhibitors OGF and retinoic acid demonstrated a greater inhibitory effect on arterial as compared with venous angiogenesis. Such differential effects on angiogenesis may be important in both defining mechanisms of action and designing therapeutic interventions.