ABSTRACT
Prime editing (PE) is a powerful gene-editing technique based on targeted gRNA-templated reverse transcription and integration of the de novo synthesized single-stranded DNA. To circumvent one of the main bottlenecks of the method, the competition of the reverse-transcribed 3' flap with the original 5' flap DNA, we generated an enhanced fluorescence-activated cell sorting reporter cell line to develop an exonuclease-enhanced PE strategy ('Exo-PE') composed of an improved PE complex and an aptamer-recruited DNA-exonuclease to remove the 5' original DNA flap. Exo-PE achieved better overall editing efficacy than the reference PE2 strategy for insertions ≥30 base pairs in several endogenous loci and cell lines while maintaining the high editing precision of PE2. By enabling the precise incorporation of larger insertions, Exo-PE complements the growing palette of different PE tools and spurs additional refinements of the PE machinery.
Subject(s)
Exonucleases , RNA, Guide, CRISPR-Cas Systems , Cell Line , DNA, Single-Stranded/genetics , Flow Cytometry , Gene Editing , CRISPR-Cas SystemsABSTRACT
Mycotoxins are secondary metabolites of fungi affecting human and animal health. Five classes of mycotoxins are of major concern in animal husbandry, namely aflatoxins, trichothecenes, zearalenone, ochratoxins, and fumonisins. Due to their diverse structure these fungal toxins are able to cause a great variety of acute symptoms in animals. Clay minerals have been used in animal nutrition to bind mycotoxins, but the binders are only very specific for aflatoxins but not for other toxins. A novel strategy to control the problem of mycotoxicoses in animals is the application of microorganisms capable of biotransforming mycotoxins into nontoxic metabolites. The microbes act in the intestinal tract of animals prior to the resorption of the mycotoxins. A Eubacterium (BBSH 797) strain is able to deactivate trichothecenes by reduction of the epoxide ring (CAST, Mycotoxins, Risks in Plant, Animal and Human Systems, Task Force Report 139, Council of Agricultural Science and Technology, Ames Iowa 2003, p. 10.; Binder, E. M., Binder, J., Ellend, N., Schaffer, E. et al., in: Miraglia, M., van Egmond, H., Brera, C., Gilbert, J. (Eds.), Mycotoxins and Phycotoxins--Developments in Chemistry, Toxicology and Food Safety, Alaken, Fort Collins 1996, pp. 279-285). This strain was isolated out of bovine rumen fluid and the mode of action was proven in vitro and also in vivo. Further a novel yeast strain, capable of degrading ochratoxin A and zearalenone was isolated and characterized (Bruinink, A., Rasonyi, T., Sidler, C., Nat. Toxins 1999, 6, 173-177; Schatzmayr, G., Heidler, D., Fuchs, E., Mohnl, M. et al., Mycotoxin Res. 2003, 19, 124-128.) Due to the yeasts affiliation to the genus of Trichosporon and its property to degrade mycotoxins this strain was named Trichosporon mycotoxinivorans (Trichosporon MTV, 115).
