The novel FLASH platform we describe here will enable any interested researcher, core facility, or institution to produce TALE repeat arrays in an inexpensive and high-throughput manner. With FLASH, DNA fragments are assembled on solid phase magnetic beads rather than in solution, thereby enabling serial enzymatic reactions to be performed without the need for column-based wash or purification steps. FLASH also avoids the need for gel isolation or analysis of intermediate constructs, both of which are labor-intensive and difficult to automate. When performed with a liquid-handling robotic platform, FLASH can be used to assemble DNA fragments encoding 96 arrays in less than a day.
Our large-scale testing of 144 FLASH-assembled TALEN pairs provides the most comprehensive test of TALEN technology performed to date. 100% of the 48 EGFP-targeted TALEN pairs and ~88% of the 96 endogenous gene-targeted TALEN pairs we produced with FLASH can cleave their targets in human cells with mutation efficiencies similar to those induced by ZFNs engineered by the selection-based OPEN method. The nucleotide composition of the 96 EGFP TALEN half-sites and the 168 endogenous gene TALEN half-sites for which we successfully made TALENs is quite diverse, reflecting DNA sequences composed of variable numbers and percentages of G, A, T, and C bases (Supplementary Figures 4
). We do not know the precise reason(s) why 12 TALEN pairs targeted to endogenous human genes failed to show activities in our T7EI assay. Possible explanations include inhibitory effects of chromatin structure or DNA modification or inefficient expression and/or folding of particular TALENs. Nonetheless, the high success rate we observe suggests that pre-screening TALENs using other surrogate assays (e.g. yeast-based reporter assays12, 17
) may be unnecessary and that TALENs might be tested directly at the endogenous gene target in the cell type or organism of interest.
We note that all of the TALENs we assembled using FLASH were made using a particular framework of TALE repeats and amino- and carboxy-terminal sequences first described by Miller and colleagues.6
This framework has been used to construct nucleases that function efficiently in nematodes,18
and human somatic6, 15, 17
and pluripotent stem cells,19
and we therefore predict that FLASH-assembled TALENs will also likely show high activities and high success rates in other cell types and organisms. A related question to test in future experiments will be whether TALENs made on any of the other various architectures described in the literature8–11, 16, 17
will also exhibit the robustness that we observe with the particular framework used in our FLASH platform.
Our results demonstrate that the targeting range of TALENs is actually substantially higher than previously suggested by the Bogdanove and Voytas groups who have described five design guidelines for choosing potential cleavage sites (Supplementary Discussion
). These guidelines limit the targeting range of TALENs to approximately one site in every 35 bps of DNA sequence17
and have been implemented in their web-based TALE-NT software17
). We found that we were able to successfully make active TALENs for 131 full target sites that fail to meet one or more of four of these design guidelines. Furthermore, we did not find any statistically significant correlation between failure to meet these four guidelines and the activity levels of our 48 EGFP-targeted TALENs. The discrepancy between these computationally-derived guidelines and our experimental results may be because the rules were derived from sites bound by monomeric TALEs whereas TALENs function as dimers.
By systematically making and testing TALENs for target sites of various lengths, we uncovered an inverse correlation between the number of TALE repeats in a TALEN and the degree of associated cytotoxicity. In our EGFP reporter experiments, we found that shorter TALENs are just as active as longer ones but these shorter TALENs also tend to be more cytotoxic, presumably due to their greater potential for binding to off-target sites elsewhere in the genome. Our findings suggest that cytotoxicity might be minimized by constructing longer TALENs (e.g.—those that harbor 14.5 to 19.5 TALE repeats), a hypothesis that can be tested in future experiments. Even with this restriction and other limitations on the length of the spacer sequence, we estimate that on average more than three TALEN pairs can be targeted per base pair of random DNA sequence (see Supplementary Discussion
Production-scale use of FLASH should enable the construction of thousands of TALEN pairs per year. We have already made a total of more than 600 TALEN-encoding plasmids using FLASH (this manuscript and data not shown). In production mode, it should be straightforward for two scientists to construct a set of 96 TALE repeat arrays at least three times per week, enabling the generation of more than 7200 TALEN pairs per year. Our cost for making a pair of sequence-verified TALEN plasmids using FLASH (including labor) is less than $200. This low per-unit cost will be particularly important for large-scale gene editing projects or for academic core facilities interested in making large numbers of nucleases. Importantly, because we have not yet fully optimized the FLASH method, we believe the cost of producing a pair of TALENs could easily be further reduced.
We have also adapted the FLASH method so that it can be performed in medium-throughput. In this modified protocol, the overall approach remains unchanged but manipulations are carried out manually using a multi-channel pipet rather than with a liquid-handling robot. We have successfully used this protocol to assemble dozens of TALE repeat arrays in one to two days (data not shown). This alternative smaller-scale protocol provides access to FLASH for laboratories who do not have automated liquid-handling equipment.
An important issue for future investigation is the extent of undesired off-target alterations introduced by TALENs. Off-target sites for one TALEN pair in the human genome have already been identified using a computational approach.19
Application of improved methods for identification of nuclease off-target cleavage events27, 28
to TALENs may reveal additional off-target sites. Whole exome or genome sequencing, as recently done with human induced pluripotent stem cells modified by ZFNs29
and with yeast modified by TALENs,16
might also be informative. All TALENs we constructed by FLASH harbor the wild-type FokI domain and therefore may form unwanted homodimers capable of inducing off-target mutations. As previously demonstrated by others, using obligate heterodimeric FokI domains may reduce formation of undesirable homodimers.28, 30–32
Until off-target sites can be comprehensively identified, users of TALENs will need to account for these undesired potential effects, just as they currently do for ZFNs.
Our large-scale demonstration of the high success rate and near limitless targeting range of TALEN technology combined with our development of the high-throughput FLASH method represent important advances for the genome engineering field. FLASH will encourage and enable any researcher to rapidly, efficiently, and precisely alter any gene or DNA sequence of interest without the need for specialized protein engineering expertise or for extensive screening to identify active nucleases. The capability of FLASH to produce TALENs in high- or medium-throughput will change the scope of gene modification experiments that can be performed by both individual laboratories and core facilities (e.g. enabling pathway- or genome-wide projects). In this regard, we note that in this study we modified more endogenous genes than any other individual report using ZFNs, meganucleases, or TALENs of the past nine years. In addition, although we have focused on TALENs in this report, FLASH should also inspire innovative applications involving the fusion of engineered TALE repeat arrays to other functional domains to create novel targetable chimeric proteins. All reagents needed to practice FLASH and all TALEN expression plasmids we assembled will be available by request to members of the academic research community (http://www.talengineering.org
). We expect that the FLASH TALEN platform should enhance the adoption and application of genome engineering technologies by a broad range of researchers.