TALE Nickase-Mediated SP110 Knockin Endows Cattle With Resistance To Tuberculosis

Tuberculosis is a zoonotic disease caused by the transmission of Mycobacterium bovis from animals to human beings and from human-to-human. Bovine tuberculosis is widely distributed worldwide, it is a serious threat to global public health and agriculture. No effective programs currently exist to eliminate or control the disease in many less-developed areas of Africa and Asia. Therefore, more extensive and effective studies on the control of bovine tuberculosis are urgently required in these regions. As previously reported that SP110 can control M.bovis growth in mouse macrophages and induce apoptosis in infected cells.And our preliminary experiments also confirmed that SP110 could strengthen the phagocytic ability of cattle macrophages. This study aims to develop TALEN technology to insert SP110 gene into the genome of Holstein–Friesian cattle, and produce transgenic cattle with ability of tuberculosis-resistance.TALEN is a programmable nuclease that can precisely cut the genome, and has been widely used to study gene function and produce transgenic animals. Numerous cases of TALEN-mediated gene knock-outs have been reported in the last two years, but successful knock-ins are rare. TALEN-mediated site-specific transgenesis has been successfully applied to model animals. However, transgenic cattle with gene knock-in have yet to be created using TALENs. This study report for the first time that TALE nickase mediated-gene knock-in apply to cattle and created 13 transgenic cattle with SP110 gene site-specific knocked-in. In vitro and in vivo challenge and transmission experiments proved that the transgenic cattle are able to control the growth and multiplication of M. bovis, turn on the apoptotic pathway of cell death instead of necrosis after infection, and efficiently resist the low dose of M. bovis transmitted from tuberculous cattle in nature. The main contents of this research are as follows:1. We designed three active TALENs specific to the intergenic region between surfactant protein A1(SFTPA1) and methionine adenosyltransferase I alpha(MAT1A) on chromosome28. The activity of TALENs in human 293-FT cells was screened with a luciferase single-strand annealing assay. The frequency of TALEN-mediated disruption at the target site in bovine fetal fibroblasts was then determined by Surveyor nuclease assays. Of the threepairs of TALENs developed, Pair 2 cleaved the target site more efficiently, as demonstrated by the increased incidence of allelic mutations.2. A mutation at the active site(D450A) that abolishes catalytic activity without affecting protein dimerization or DNA recognition was introduced to FokI of the right hand of TALENs.Then in vitro DNA cleavage assay confirmed that TALE nickase-mediated single-strand break had the potential to restrict repair to the homology-directed repair pathway.3. We isolated 26 heterozygous colonies with SP110 site-specific knockin by junction PCR screening and Southern blot analysis. Using these cell colonies as donor cells, we produced 13 transgenic cattle with SP110 site-specific knockin by somatic cell nuclear transfer.4. To estimate the ability of SP110 transgenic cattle to respond to M. bovis infection,macrophages from peripheral blood were challenged with M. bovis in vitro. The result indicated that the transgenic cattle were able to control the growth and multiplication of M.bovis. We also observed a clear distinction in the mechanism of macrophage cell death after infection by flow cytometry. The control cattle macrophages showed characteristic necrosis,whereas the transgenic cattle macrophages showed remarkable apoptosis. In vivo challenge experiment was also performed by endobronchial instillation of M. bovis. Cattle were killed for postmortem examination. The result showed that the gross pathology score and CFU count of transgenic cattle were significantly lower than that of the control cattle. To further estimate the tuberculosis-resistant ability of transgenic cattle, a transmission experiment was also performed by feeding transgenic cattle with tuberculosis cattle in a con?ned airspace. The tuberculin skin tests, gamma interferon release assays and MTB-specific enzyme-linked immunospot assays confirmed that the SP110 transgenic cattle were able to resist the infection of M.bovis. Besides, postmortem examination was also performed, and the gross pathology score of transgenic cattle were significantly lower than that of the control cattle.5. We also designed three active TALENs specific to the intergenic region between fascin homolog 1(FSCN1) and beta actin(ACTB) genes. We identified eight putative off-target sites of the M-S locus and F-A locus respectively by sequence similarity using comparison with bovine genome. Of the 22 M-S locus gene-targeted cell clones and 19 F-A locus gene-targeted cell clones, we detected one off-target event. Although TALE nickases can be used for genetic manipulation in the bovine genome at any locus, there are still chances to led unpredictable damages to cells. Therefore, we should look for a "safe harbor" before gene knockin manipulation using TALENs.Here we report, for the first time to our knowledge, TALE nickase-mediated gene insertion via homologous recombination to produce transgenic cattle with increased resistanceto tuberculosis. Our results contribute to the control and prevention of bovine tuberculosis and provide a new insight into breeding animals for disease resistance.