RESUMO
Nucleic acid detection by isothermal amplification and the collateral cleavage of reporter molecules by CRISPR-associated enzymes is a promising alternative to quantitative PCR. Here, we report the clinical validation of the specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) assay using the enzyme Cas13a from Leptotrichia wadei for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the virus that causes coronavirus disease 2019 (COVID-19)-in 154 nasopharyngeal and throat swab samples collected at Siriraj Hospital, Thailand. Within a detection limit of 42 RNA copies per reaction, SHERLOCK was 100% specific and 100% sensitive with a fluorescence readout, and 100% specific and 97% sensitive with a lateral-flow readout. For the full range of viral load in the clinical samples, the fluorescence readout was 100% specific and 96% sensitive. For 380 SARS-CoV-2-negative pre-operative samples from patients undergoing surgery, SHERLOCK was in 100% agreement with quantitative PCR with reverse transcription. The assay, which we show is amenable to multiplexed detection in a single lateral-flow strip incorporating an internal control for ribonuclease contamination, should facilitate SARS-CoV-2 detection in settings with limited resources.
Assuntos
COVID-19/diagnóstico , Proteínas Associadas a CRISPR/genética , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/genética , SARS-CoV-2/genética , COVID-19/virologia , Humanos , Leptotrichia/enzimologia , Pandemias/prevenção & controleRESUMO
Programmable RNA editing enables reversible recoding of RNA information for research and disease treatment. Previously, we developed a programmable adenosine-to-inosine (A-to-I) RNA editing approach by fusing catalytically inactivate RNA-targeting CRISPR-Cas13 (dCas13) with the adenine deaminase domain of ADAR2. Here, we report a cytidine-to-uridine (C-to-U) RNA editor, referred to as RNA Editing for Specific C-to-U Exchange (RESCUE), by directly evolving ADAR2 into a cytidine deaminase. RESCUE doubles the number of mutations targetable by RNA editing and enables modulation of phosphosignaling-relevant residues. We apply RESCUE to drive ß-catenin activation and cellular growth. Furthermore, RESCUE retains A-to-I editing activity, enabling multiplexed C-to-U and A-to-I editing through the use of tailored guide RNAs.
Assuntos
Adenosina Desaminase/genética , Citidina/genética , Citosina Desaminase/genética , Engenharia de Proteínas/métodos , Edição de RNA , Proteínas de Ligação a RNA/genética , Uridina/genética , Adenosina/genética , Adenosina Desaminase/química , Citosina Desaminase/química , Células HEK293 , Humanos , Inosina/genética , Domínios Proteicos , Proteínas de Ligação a RNA/química , beta Catenina/química , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Brain metastases are an increasingly important determinant of survival and quality of life in patients with cancer. Current approaches to the management of brain metastases are driven by prognostic factors, including the Karnofsky Performance Status, tumor histology, number of metastases, patient age, and status of systemic disease. Most brain metastases are treated with radiosurgery, computer-assisted surgery, or whole brain radiation therapy. Remarkable advances in computer-assisted neuronavigation have made neurosurgical removal of metastases safer, even in eloquent areas of the brain. Computerization also enhances the efficacy and safety of conformal radiosurgery planning using various modern stereotactic radiosurgery (SRS) technologies, including newer frameless-based systems. Controversial issues include whether to defer whole brain radiotherapy (WBRT) in patients undergoing SRS or image-guided surgery and when to use SRS "boost" in a patient undergoing WBRT. The determination of how best to apply these treatments for individual patients cannot be standardized to a single paradigm, but data from well-controlled studies help physicians make informed decisions about the benefits and risks of each approach.