TALE nickase-mediated SP110 knockin endows cattle with increased resistance to tuberculosis Haibo Wua,b, Yongsheng Wanga,b, Yan Zhanga,b, Mingqi Yanga, Jiaxing Lvb, Jun Liua,b, and Yong Zhanga,b,1 aCollege of Veterinary Medicine and bKey Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling 712100, Shaanxi, China Edited by Leif Andersson, Uppsala University, Uppsala, Sweden, and approved February 3, 2015 (received for review November 11, 2014) Transcription activator-like effector nuclease (TALEN)-mediated ge- livestock species. Moreover, the results of the present study nome modification has been applied successfully to create transgenic could contribute to the control of tuberculosis. animals in various species, such as mouse, pig, and even monkey. However, transgenic cattle with gene knockin have yet to be created Results using TALENs. Here, we report site-specific knockin of the transcrip- Construction of TALEN Plasmids and Activity Assessment. In con- tion activator-like effector (TALE) nickase-mediated SP110 nuclear sideration of potential synergistic effects of neighboring genes, body protein gene (SP110) via homologous recombination to pro- we designed three active TALENs specific to the intergenic re- duce tuberculosis-resistant cattle. In vitro and in vivo challenge gion between surfactant protein A1 (SFTPA1) and methionine and transmission experiments proved that the transgenic cattle adenosyltransferase I alpha (MAT1A) on chromosome 28 (Fig. are able to control the growth and multiplication of Mycobacterium 1A and SI Appendix, Table S1). Because the transfection effi- bovis, turn on the apoptotic pathway of cell death instead of necrosis ciency of TALEN plasmid and mRNA into bovine fetal fibro- after infection, and efficiently resist the low dose of M. bovis trans- blasts (BFFs) is extremely low, EGFP or mCherry was added to mitted from tuberculous cattle in nature. In this study, we developed the TALEN vectors with a self-cleaving T2A peptide to sort TALE nickases to modify thegenomeofHolstein–Friesian cattle, transfected cells via flow cytometry (Fig. 1B). thereby engineering a heritable genome modification that facil- The activity of TALENs in human 293-FT cells was screened itates resistance to tuberculosis. with a luciferase single-strand annealing (SSA) assay [pair 1 (9.2 ± 0.86) vs. pair 2 (35.8 ± 3.75), P = 0.000; pair 1 (9.2 ± 0.86) vs. TALEN | homologous recombination | single-strand break | tuberculosis | pair 3 (39.7 ± 3.17), P = 0.000; pair 2 (35.8 ± 3.75) vs. pair 3 (39.7 disease resistance ± 3.17), no significance] (Fig. 1C). The frequency of TALEN- mediated disruption at the target site in BFFs then was determined ene targeting by homologous recombination can modify the by Surveyor nuclease assays (27). Of the three pairs of TALENs Ggenome precisely and has been widely used to study gene developed, pair 2 cleaved the target site most efficiently, as function and produce transgenic animals (1–5). Transcription demonstrated by the increased incidence of allelic mutations activator-like effector nuclease (TALEN) is a programmable nu- (NHEJ frequency) (Fig. 1 D and E). Therefore, pair 2 was used clease that contains a FokI nuclease domain and a DNA-binding for subsequent experiments. To confirm further the presence of “ ” domain known as transcription activator-like effector derived nuclease-induced insertions and deletions at the targeted locus, from the plant pathogenic bacteria Xanthomonas spp. TALEN the targeted locus was PCR amplified from the genomic DNA induces a double-strand break (DSB) at a precise, defined po- and transformed into Escherichia coli by TA cloning. We ran- sition in the genome, resulting in unpredictable gene mutations domly picked 865 bacterial colonies for sequencing. Deletions or when the DSBs are repaired erroneously by nonhomologous end insertions were detected in 6.13% of the colonies generated from joining (NHEJ) (6, 7). However, TALENs also can be used in conjunction with specially designed exogenous donor DNA to Significance generate large-scale deletions, gene disruptions, DNA additions, or single-nucleotide changes (8, 9). Numerous cases of TALEN- mediated gene knockouts have been reported in the last 2 y (9– Bovine tuberculosis is a chronic infectious disease that affects 12), but successful knockins are rare (7, 13). TALEN-mediated a broad range of mammalian hosts. It is a serious threat to site-specific transgenesis has been applied successfully to model agriculture in many less-developed countries. In this study, we animals (7, 14–16) and even in large livestock, such as pigs and introduced a mutation to the FokI of the right hand of wild- cattle (17–21). However, to the best of our knowledge, transgenic type transcription activator-like effector nuclease and estab- cattle with gene knockin have yet to be created using TALENs (19). lished a transcription activator-like effector nickase system that Tuberculosis is a zoonotic disease caused by the transmission creates single-strand breaks in the genome. Then we used this SP110 of Mycobacterium bovis from animals to human beings and from system to add the mouse gene toaspecificlocationinthe human to human (22). It is a serious threat to global public bovine genome and created transgenic cattle with increased re- health and agriculture (23, 24). Bovine tuberculosis is widely sistance to tuberculosis. Our results contribute to the control and distributed worldwide, and no effective programs currently exist prevention of bovine tuberculosis and provide a previously un- identified insight into breeding animals for disease resistance. to eliminate or control the disease in many less-developed areas of Africa and Asia (24, 25). Therefore more extensive and ef- Author contributions: H.W. and Yong Zhang designed research; H.W., Y.W., Yan Zhang, fective studies on the control of bovine tuberculosis are urgently M.Y., J. Lv, and J. Liu performed research; H.W. and Yong Zhang analyzed data; and H.W. required in these regions. The mouse SP110 gene is emerging as wrote the paper. a promising candidate in the control of Mycobacterium tubercu- The authors declare no conflict of interest. losis (MTB) infections (26). SP110 can control MTB growth in This article is a PNAS Direct Submission. macrophages and induce apoptosis in infected cells. In this study, Freely available online through the PNAS open access option. we developed transcription activator-like effector (TALE) nick- See Commentary on page 3854. ase technology to insert a mouse SP110 gene into the genome of 1To whom correspondence should be addressed. Email: [email protected]. – Holstein Friesian cattle. Therefore, TALEN represents a vali- This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. dated tool for the targeted genetic modification of this important 1073/pnas.1421587112/-/DCSupplemental. E1530–E1539 | PNAS | Published online March 2, 2015 www.pnas.org/cgi/doi/10.1073/pnas.1421587112 Downloaded by guest on October 1, 2021 PNAS PLUS SEE COMMENTARY Fig. 1. Activity assessment of TALENs. (A) Schematic representation of the targeting locus. (B) Schematic representation of the TALEN constructs. The lengths of constructs were calculated based on TALEN-encoding plasmids with 17 modules. (C) Cleavage activity of each TALEN was measured by luciferase SSA assay in human 293-FT cells. Data are presented as mean ± SD and are derived from three independent experiments. **P < 0.01. (D) Frequency of allelic mutation as determined by Surveyor nuclease assays. Different amounts of each TALEN transfected are shown as indicated. (E)TheM-S locus was PCR amplified, and cleavage of the locus was measured using a Surveyor nuclease assay. The degree of cleavage was quantified and is shown below each lane. (F) Some of the representative sequences revealed distinct TALEN-induced insertions and deletions at the targeted locus. The binding site of TALEN is underlined in red. Occurrences of deletions and inser- tions are listed on the right. The lowercase letters rep- resent inserted bases. TALEN-transfected cells (SI Appendix, Table S2; representative Then we further confirmed that a TALE nickase-mediated sequences are shown in Fig. 1F). SSB had the potential to restrict repair to the HDR pathway. Wild-type TALEN or TALE nickase was transfected into BFFs, TALE Nickase Restricts Repair to the Homology-Directed Repair Pathway. and genomic DNA was extracted after 72 h. Surveyor nuclease A targeted single-strand break (SSB) has the potential to restrict assays were performed. As shown in Fig. 2C, TALE nickase repair to the homology-directed repair (HDR) pathway (28, 29). dramatically decreased the DNA-cleaving ability, but no NHEJ Zinc-finger nickases (ZFNs) are well established for generating events were detected. The targeted locus then was PCR ampli- SCIENCES SSBs; more recently, this strategy even has been reported in AGRICULTURAL – fied and transformed into E. coli, and 823 bacterial colonies were TALENs (5, 29 32). Therefore, a mutation at the active site picked and sent for sequencing. Compared with the 6.13% of (D450A) that abolishes catalytic activity without affecting pro- deletions or insertions in the wild-type TALEN group, none was tein dimerization or DNA recognition was introduced to FokI of the right hand of TALENs (28). An in vitro DNA cleavage assay was performed to assess the activity of TALENs bearing the D450A mutation. A linear 383-bp PCR fragment containing an off-center target site for specific TALENs was digested with in vitro-synthesized TALENs. The expected digestion patterns following strand-specific cleavage when resolved under nondenaturing and denaturing conditions are shown in Fig. 2A. Thus, provision of wild-type TALEN synthesized in vitro resulted in efficient double-strand cleavage of the template DNA, regardless of whether the products were resolved under nondenaturing or denaturing conditions (Fig. 2B, lanes 1 and 3, >71% cleavage efficiency). In contrast, introduc- tion of D450A to the wild-type TALEN (TALE nickase) elimi- nated double-strand cleavage (Fig. 2B, lane 2).