Clarke R, Merrill BJ, MacDougall MS, Pennington HM, Shy BR.. "United States Patent WO2019067322 CRISPR/CAS SYSTEM AND METHOD FOR GENOME EDITING AND MODULATING TRANSCRIPTION.", University of Illinois
Infant in extremis: respiratory failure secondary to lower airway infantile hemangioma.
2022; 22 (1): 744
Infantile hemangiomas (IHs) are vascular tumors that commonly affect infants and usually regress spontaneously or can be easily treated as an outpatient with topical beta-blockers. However, IHs that present in the airway may cause life-threatening symptoms due to airway obstruction or risk of bleeding. Here we present the first documented case of an infant with rapid deterioration and acute respiratory failure secondary to a lower airway hemangioma.This 3-month-old male initially presented in respiratory distress with symptoms consistent with a viral respiratory infection, however showed no clinical improvement with standard therapies. An urgent CT scan revealed a mass occluding the right mainstem bronchus. Upon transfer to a tertiary care facility, he developed acute respiratory failure requiring emergent intubation and single lung ventilation. The availability of multiple subspecialists allowed for stabilization of a critically ill child, expedited diagnosis, and ultimately initiation of life-saving treatment with beta blockers. After 17 total hospital days, he was extubated successfully and discharged home in good condition.While IH is a rare cause of infantile respiratory distress, we present multiple pearls for the general pediatrician for management of IHs of the airway.
View details for DOI 10.1186/s12887-022-03821-1
View details for PubMedID 36581920
Sequential Activation of Guide RNAs to Enable Successive CRISPR-Cas9 Activities
2021; 81 (2): 226-+
Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.
View details for DOI 10.1016/j.molcel.2020.12.003
View details for Web of Science ID 000672600500005
View details for PubMedID 33378644
Intracellular Ca2+ Homeostasis and Nuclear Export Mediate Exit from Naive Pluripotency
CELL STEM CELL
2019; 25 (2): 210-+
Progression through states of pluripotency is required for cells in early mammalian embryos to transition away from heightened self-renewal and toward competency for lineage specification. Here, we use a CRISPR mutagenesis screen in mouse embryonic stem cells (ESCs) to identify unexpected roles for nuclear export and intracellular Ca2+ homeostasis during the exit out of the naive state of pluripotency. Mutation of a plasma membrane Ca2+ pump encoded by Atp2b1 increased intracellular Ca2+ such that it overcame effects of intracellular Ca2+ reduction, which is required for naive exit. Persistent self-renewal of ESCs was supported both in Atp2b1-/-Tcf7l1-/- double-knockout ESCs passaged in defined media alone (no LIF or inhibitors) and in wild-type cells passaged in media containing only calcitonin and a GSK3 inhibitor. These new findings suggest a central role for intracellular Ca2+ in safeguarding naive pluripotency.
View details for DOI 10.1016/j.stem.2019.04.015
View details for Web of Science ID 000478084700008
View details for PubMedID 31104942
View details for PubMedCentralID PMC6685429
Enhanced Bacterial Immunity and Mammalian Genome Editing via RNA-Polymerase-Mediated Dislodging of Cas9 from Double-Strand DNA Breaks
2018; 71 (1): 42-+
The ability to target the Cas9 nuclease to DNA sequences via Watson-Crick base pairing with a single guide RNA (sgRNA) has provided a dynamic tool for genome editing and an essential component of adaptive immune systems in bacteria. After generating a double-stranded break (DSB), Cas9 remains stably bound to DNA. Here, we show persistent Cas9 binding blocks access to the DSB by repair enzymes, reducing genome editing efficiency. Cas9 can be dislodged by translocating RNA polymerases, but only if the polymerase approaches from one direction toward the Cas9-DSB complex. By exploiting these RNA-polymerase/Cas9 interactions, Cas9 can be conditionally converted into a multi-turnover nuclease, mediating increased mutagenesis frequencies in mammalian cells and enhancing bacterial immunity to bacteriophages. These consequences of a stable Cas9-DSB complex provide insights into the evolution of protospacer adjacent motif (PAM) sequences and a simple method of improving selection of highly active sgRNAs for genome editing.
View details for DOI 10.1016/j.molcel.2018.06.005
View details for Web of Science ID 000438142200007
View details for PubMedID 29979968
View details for PubMedCentralID PMC6063522
Co-incident insertion enables high efficiency genome engineering in mouse embryonic stem cells
NUCLEIC ACIDS RESEARCH
2016; 44 (16): 7997-8010
CRISPR/Cas9 nucleases have enabled powerful, new genome editing capabilities; however, the preponderance of non-homologous end joining (NHEJ) mediated repair events over homology directed repair (HDR) in most cell types limits the ability to engineer precise changes in mammalian genomes. Here, we increase the efficiency of isolating precise HDR-mediated events in mouse embryonic stem (ES) cells by more than 20-fold through the use of co-incidental insertion (COIN) of independent donor DNA sequences. Analysis of on:off-target frequencies at the Lef1 gene revealed that bi-allelic insertion of a PGK-Neo cassette occurred more frequently than expected. Using various selection cassettes targeting multiple loci, we show that the insertion of a selectable marker at one control site frequently coincided with an insertion at an unlinked, independently targeted site, suggesting enrichment of a sub-population of HDR-proficient cells. When individual cell events were tracked using flow cytometry and fluorescent protein markers, individual cells frequently performed either a homology-dependent insertion event or a homology-independent event, but rarely both types of insertions in a single cell. Thus, when HDR-dependent selection donors are used, COIN enriches for HDR-proficient cells among heterogeneous cell populations. When combined with a self-excising selection cassette, COIN provides highly efficient and scarless genome editing.
View details for DOI 10.1093/nar/gkw685
View details for Web of Science ID 000384687000043
View details for PubMedID 27484482
View details for PubMedCentralID PMC5027516
Topical Vapocoolant-Associated Vaso-occlusive Event in a 10-year-old with Sickle Cell Disease.
Pain management nursing : official journal of the American Society of Pain Management Nurses
2021; 22 (5): 631-633
Vapocoolant sprays are convenient forms of cold temperature analgesia. These sprays may not be suitable for all patients with particular concern for patients with sickle cell disease. To prevent any further cases from occurring, we propose adding a more specific cautionary statement to the manufacturer guidelines. We also hope that medical personnel can help patients with sickle cell avoid topical and environmental cold temperature triggers for sickle vaso-occlusive pain and reduce the suffering in this rare disease.
View details for DOI 10.1016/j.pmn.2021.04.008
View details for PubMedID 34090800
The NAMPT Promoter Is Regulated by Mechanical Stress, Signal Transducer and Activator of Transcription 5, and Acute Respiratory Distress Syndrome-Associated Genetic Variants
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY
2014; 51 (5): 660-667
Increased nicotinamide phosphoribosyltransferase (NAMPT) transcription is mechanistically linked to ventilator-induced inflammatory lung injury (VILI), with VILI severity attenuated by reduced NAMPT bioavailability. The molecular mechanisms of NAMPT promoter regulation in response to excessive mechanical stress remain poorly understood. The objective of this study was to define the contribution of specific transcription factors, acute respiratory distress syndrome (ARDS)-associated single nucleotide polymorphisms (SNPs), and promoter demethylation to NAMPT transcriptional regulation in response to mechanical stress. In vivo NAMPT protein expression levels were examined in mice exposed to high tidal volume mechanical ventilation. In vitro NAMPT expression levels were examined in human pulmonary artery endothelial cells exposed to 5 or 18% cyclic stretch (CS), with NAMPT promoter activity assessed using NAMPT promoter luciferase reporter constructs with a series of nested deletions. In vitro NAMPT transcriptional regulation was further characterized by measuring luciferase activity, DNA demethylation, and chromatin immunoprecipitation. VILI-challenged mice exhibited significantly increased NAMPT expression in bronchoalveolar lavage leukocytes and in lung endothelium. A mechanical stress-inducible region (MSIR) was identified in the NAMPT promoter from -2,428 to -2,128 bp. This MSIR regulates NAMPT promoter activity, mRNA expression, and signal transducer and activator of transcription 5 (STAT5) binding, which is significantly increased by 18% CS. In addition, NAMPT promoter activity was increased by pharmacologic promoter demethylation and inhibited by STAT5 silencing. ARDS-associated NAMPT promoter SNPs rs59744560 (-948G/T) and rs7789066 (-2,422A/G) each significantly elevated NAMPT promoter activity in response to 18% CS in a STAT5-dependent manner. Our results show that NAMPT is a key novel ARDS therapeutic target and candidate gene with genetic/epigenetic transcriptional regulation in response to excessive mechanical stress.
View details for DOI 10.1165/rcmb.2014-0117OC
View details for Web of Science ID 000347419400007
View details for PubMedID 24821571