title: Insertion of Group II Intron-Based Ribozyme Switches into Homing Endonuclease Genes.
abstract: Fungal mitochondrial genomes act as "reservoirs" for homing endonucleases. These enzymes with their DNA site-specific cleavage activities are attractive tools for genome editing, targeted mutagenesis and gene therapy applications. Herein, we present strategies where homing endonuclease open reading frames (HEases ORFs) are interrupted with group II intron sequences. The ultimate goal is to achieve in vivo expression of HEases that can be regulated by manipulating the splicing efficiency of the HEase ORF-embedded group II introns. That addition of exogenous magnesium chloride (MgCl2) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl2) to the growth medium antagonizes the expression of HEase activity (i.e., splicing). Group II introns are potentially autocatalytic self-splicing elements and thus can be used as molecular switches that allow for temporal regulated HEase expression. This should be useful in precision genome engineering, mutagenesis, and minimizing off-target activities.
authors.affiliation: Department of Microbiology, 401 University of Manitoba, Buller Building 213, Winnipeg, MB, Canada, R3T 2N2.
authors.forename: Tuhin Kumar
authors.initials: TK
authors.lastname: Guha
authors.affiliation: Department of Microbiology, 401 University of Manitoba, Buller Building 213, Winnipeg, MB, Canada, R3T 2N2. hausnerg@cc.umanitoba.ca.
authors.forename: Georg
authors.initials: G
authors.lastname: Hausner
mesh_terms: D004272:DNA, Mitochondrial / Q000235:genetics; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D007438:Introns / Q000235:genetics*; D016366:Open Reading Frames / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics*
chemical_list: D004272:DNA, Mitochondrial; D016337:RNA, Catalytic; D004720:Endonucleases
keywords: Biotechnology; Group II introns; Homing endonuclease assay; Homing endonucleases; Targeted mutagenesis; Temporal regulation