ABSTRACT
CRISPR-Cas systems have revolutionized genome editing across a broad range of biotechnological endeavors. Many CRISPR-Cas nucleases have been identified and engineered for improved capabilities. Given the modular structure of such enzymes, we hypothesized that engineering chimeric sequences would generate non-natural variants that span the kinetic parameter landscape, and thus provide for the rapid selection of nucleases fit for a particular editing system. Here, we design a chimeric Cas12a-type library with approximately 560 synthetic chimeras, and select several functional variants. We demonstrate that certain nuclease domains can be recombined across distantly related nuclease templates to produce variants that function in bacteria, yeast, and human cell lines. We further characterize selected chimeric nucleases and find that they have different protospacer adjacent motif (PAM) preferences and the M44 chimera has higher specificity relative to wild-type (WT) sequences. This demonstration opens up the possibility of generating nuclease sequences with implications across biotechnology.
Subject(s)
CRISPR-Cas Systems , Endonucleases/metabolism , Gene Editing/methods , Recombinant Fusion Proteins/metabolism , Bacteria/genetics , Biotechnology/methods , Endonucleases/genetics , Gene Library , HEK293 Cells , Humans , Mutation , Recombinant Fusion Proteins/genetics , Reproducibility of Results , Yeasts/geneticsABSTRACT
The Suzuki coupling reaction of basic nitrogen containing substrates (2-bromo- and 2-chloro-4-aminopyridine, and 2-bromo and 2-chloropyridine) with phenylboronic acid using Pd(TPP)2Cl2/K3PO4 in acetonitrile-water biphasic solvent systems under a CO2 or a N2 atmosphere is discussed. It was observed that 2-halo-4-aminopyridine produced quantitative yields of coupled products under a CO2 atmosphere while the yields for the 2-halopyridines were poor. In contrast, the yields of coupled products for the 2-halopyridines substrates were quantitative under a N2 atmosphere while only poor yields were realized for the 2-halo-4-aminopyridines under the same conditions. Evidence is presented which suggests that the presence of CO2 alters the pH of the aqueous phase of the reaction system and the accompanying efficiency of the coupling process. Using a series of buffers to adjust the pH of the aqueous phase, the pH dependence associated with the efficiency of the coupling process is illustrated.
Subject(s)
Carbon Dioxide/chemistry , Nitrogen/chemistry , Pressure , Acetonitriles/chemistry , Hydrogen-Ion Concentration , Kinetics , Phosphates/chemistry , Phosphines/chemistry , Potassium Compounds/chemistry , TemperatureABSTRACT
The properties and uses of piperylene sulfone as a new, recyclable dipolar, aprotic solvent for conducting organic reactions are presented.
ABSTRACT
The journey to holism is the result of a grounded theory study of 18 members of the American Holistic Nurses' Association. Participants were prompted to "Tell me about holistic nursing" and replies revealed a seven-step process to becoming a holistic nurse. The process is an expansion of Van Gennep's three-stage theory of rites of passage, which involves separation, marginality/liminality, and reintegration. The expanded process begins with separation from mainstream nursing and concludes with reintegration into nursing as a holistic nurse. The marginality/liminality step shows five active steps: gathering information to quell the distress of the separation, applying that information to others, changing focus to self-care, applying the information to self, and increasing self-knowledge. This article describes the process and presents data to support the conclusion that the growth of a holistic nurse follows a recognizable course.
