A robust TALENs system for highly efficient mammalian genome editing

Loss of function (LOF) is a powerful approach in the study of gene function. To achieve this, numerous technologies have been developed, such as RNA interference to downregulate the RNA transcripts of target genes. Recently, site-specific nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), have been used to disrupt target gene function by creating DNA insertions or mutations1,2. Since ZFNs or TALENs can completely abolish the function of targeted genes, when used in LOF studies, the results are much cleaner and easier to interpret than with RNA interference. In the past 15 years, significant effort has been invested in ZFNs, in which the DNA-binding property of the zinc finger domain of a class of transcription factors is exploited to bind specifically to the intended genomic sequence3. However, due to the complexity of the zinc finger domain's DNA-binding code, significant effort and expertise in the design and optimization of ZFN activity and specificity are usually required, preventing the wide adoption of ZFNs to date. Recently, TALENs have emerged as a breakthrough technology for genomic editing1, allowing precise gene mutation or deletion at almost any specific genomic locus. The TALE protein consists of a translocation domain in the N-terminal, a nuclear localization signal in the C-terminal, and various tandems of 33–34 amino acid repeats in the middle for target DNA sequence detection and binding1. Each tandem repeat has two variable amino acid residues at positions 12 and 13, designated repeat variable diresidues (RVDs)4. The RVDs NI, NG, HD, and NN preferentially recognize adenine (A), thymine (T), cytosine (C), and guanine (G), respectively (Fig. 1A)5,6. When fused with the nonspecific FokI endonuclease catalytic domain at the C-terminal, TALENs form dimers through binding to two target sequences separated by ~17 bases. Between the pair of binding sites, the FokI catalytic domains dimerize and function as molecular scissors by introducing double-strand breaks (DSBs). The resulting DSBs are usually repaired by the error-prone non-homologous end-joining pathway (NHEJ), frequently leading to small DNA deletion or insertion and consequent frameshift in the open reading frames (ORFs) of targeted genes, and thus, functional gene knockout1. Figure 1 Our improved TALENs system. Due to their modular design, ease of use, high efficiency, low off-target rate, and capacity to precisely target almost any genomic locus1, TALENs are superior to ZFNs for genomic editing. TALENs are also highly effective tool in introducing mutations into an endogenous genome, making the study of gene mutants in an endogenous physiological setting much easier7. Furthermore, when mRNAs encoding TALENs are directly injected into the cytoplasm of one-cell-stage embryos, homozygous gene mutation or knockout can be achieved with a relatively high efficiency8, saving months, even years of work compared to more traditional mouse genetics approaches. When establishing a cell line with somatic gene knockout or knockin, two plasmids that encode a pair of TALENs need to be co-transfected into cells. The cells are then diluted to isolate clones with the intended genomic editing. Usually, a large number of cell clones need to be screened to select cells with successful gene disruption, especially in hard-to-transfect cells. In addition, because mammalian cells usually contain two copies of each chromosome, homozygous gene editing requires that both alleles be targeted. Due to genomic instability, aneuploidy is frequent in cancer cells9, making homozygous genomic editing even more challenging. To improve TALEN targeting efficiency in these cells, a more robust TALENs system is clearly needed. Recently, a strategy was reported by co-transfecting a surrogate reporter, in which a piece of DNA that is homologous to the intended ZFNs or TALENs target sequence is cloned between a RFP and GFP gene, allowing enriching cells with nuclease-induced mutations by isolating dual-labelled cells with fluorescence-activated cell sorting (FACS)10. However, a major caveat of this approach is that distinct surrogate reporters are required for every gene targeting experiment, especially when multiple genes need to be targeted or multiple designs of TALENs targeting the same gene need to be tested. To establish a simple and efficient TALENs system, we introduced genes that encode different fluorescent proteins into the TALEN-encoding plasmids. No additional work is required for this system other than essential TALENs cloning for genomic editing experiment. After co-transfection, TALENs-transfected cells can be enriched by isolating dual-labelled cells by FACS, greatly improving the efficiency of genomic editing. We also provide a practical guide on the use of this TALEN system for laboratories without prior experience with this powerful new technology.