Guha Tuhin Kumar, Hausner Georg
Department of Microbiology, 401 University of Manitoba, Buller Building 213, Winnipeg, MB, Canada, R3T 2N2.
Methods Mol Biol. 2017;1498:135-152. doi: 10.1007/978-1-4939-6472-7_9.
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 (MgCl) appears to stimulate splicing of nonnative group II introns in Escherichia coli and the addition of cobalt chloride (CoCl) 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.
真菌线粒体基因组充当归巢内切酶的“储存库”。这些具有DNA位点特异性切割活性的酶是基因组编辑、靶向诱变和基因治疗应用中极具吸引力的工具。在此，我们提出了一些策略，其中归巢内切酶开放阅读框（HEases ORFs）被II类内含子序列中断。最终目标是实现HEases的体内表达，其可通过操纵嵌入HEase ORF的II类内含子的剪接效率来调节。添加外源氯化镁（MgCl）似乎能刺激大肠杆菌中非天然II类内含子的剪接，而向生长培养基中添加氯化钴（CoCl）会拮抗HEase活性（即剪接）的表达。II类内含子是潜在的自催化自我剪接元件，因此可作为分子开关，实现对HEase表达的时间调控。这在精确基因组工程、诱变以及最小化脱靶活性方面应该会很有用。
