ABSTRACT
The discovery of CRISPR-Cas9 has revolutionized molecular biology, greatly accelerating the introduction of genetic modifications into organisms and facilitating the development of novel therapeutics and diagnostics. For many applications, guide RNA and Cas9 protein are expressed, combined, and purified to produce a ribonucleic enzyme complex that is then added into a diagnostic device or delivered into cells. The objective of this work was to develop an ultrafiltration process for the selective purification of Cas9 ribonucleoprotein by removal of excess guide RNA. A His-tagged Streptococcus pyogenes Cas9 protein was produced in Escherichia coli, purified by metal affinity chromatography, and complexed with a 40 kDa (124 nucleotide) single guide RNA. Ultrafiltration experiments were first performed on solutions containing either guide RNA or Cas9 protein to identify the effect of filtration conditions and membrane pore size on the selectivity. Shear-induced aggregation of the Cas9 led to significant fouling under some conditions. A diafiltration process was then developed using a Biomax® 300 kDa polyethersulfone membrane to selectively remove excess guide RNA from a solution containing Cas9-bound guide RNA and free guide RNA. These results demonstrate the potential of using ultrafiltration for the removal of excess RNA during the production of functional ribonucleoprotein complexes.
Subject(s)
CRISPR-Associated Protein 9/isolation & purification , Chromatography, Affinity/methods , Escherichia coli/metabolism , Histidine/chemistry , RNA, Guide, Kinetoplastida/isolation & purification , Streptococcus pyogenes/enzymology , Ultrafiltration/methods , CRISPR-Associated Protein 9/chemistry , CRISPR-Associated Protein 9/metabolism , Escherichia coli/genetics , Streptococcus pyogenes/geneticsABSTRACT
It is well established that the structure of plasmid DNA is a strong function of solution ionic conditions due to changes in intramolecular electrostatic interactions between the charged phosphate groups along the DNA backbone. Multivalent cations like spermine and spermidine play a critical role in compacting and controlling the structure of supercoiled DNA in living cells. The objective of this work was to investigate the effects of these polyamines on the ultrafiltration of plasmid DNA, including possible opportunities to use these polycations to enhance the purification of specific plasmid isoforms. Data were obtained using a wide range of spermine and spermidine concentrations to evaluate DNA transmission through Biomax polyethersulfone ultrafiltration membranes. Spermine has a very strong effect on DNA transmission, with the sieving coefficient of the supercoiled plasmid decreasing by more than an order of magnitude upon addition of only 15 µM spermine. A comparable change in DNA transmission required >300 µM of the trivalent spermidine. The polyamines were able to significantly increase the selectivity for the separation of DNA from a model protein, but they were unable to provide a significant increase in the selectivity for separating DNA isoforms under the conditions examined in this study. The results do demonstrate that both spermine and spermidine can be used to control the extent of DNA transmission/purification during ultrafiltration. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2765, 2019.
Subject(s)
DNA/chemistry , Plasmids/chemistry , Polyamines/chemistry , DNA/isolation & purification , Plasmids/isolation & purification , UltrafiltrationABSTRACT
Se analizaron los resultados de 248 histerosalpingografías practicadas en la evalución de igual número de mujeres que consultaron por esterilidad e infertilidad. La patología tubaria fue el hallazgo más frecuente, siendo la exclusión tubárica (18,2%) la anormalidad de mayor incidencia. Las anomalías congénitas del útero representan el 5,2%, mientras que el pólipo mucoso (11,3%) y la hiperplasia endometrial fueron los hallazgos que más se observaron en la patología corporal. Por la alta incidencia de imágenes patológicas *82,5%), su fácil y segura ejecución, la HSG debe ser exploración rutinaria en pacientes infértiles
