Many years of effort spent in elucidating the interaction between TAL effectors and their modulated host genes has led to a recent breakthrough in deciphering the DNA recognition code of TAL effectors (28
). The predictability and potential manipulability of the TAL central repeat domain for DNA-binding specificities make TAL an excellent system for exploiting potential biotechnological applications. In the present study, we created chimeric TALNs, FN-AvrXa7, AvrXa7-FN and PthXo1-FN containing the entire AvrXa7 or PthXo1 TAL effectors and the nuclease domain of the FokI restriction enzyme either at the C-terminal end or the N-terminal end of each TAL effector. All three constructs were tested for the ability to bind to the respective EBE recognition site and to cleave adjacent DNA. Binding of FN-AvrXa7 to the AvrXa7 EBE in vivo
was demonstrated by its ability to activate transcription of a GFP coding sequence driven by the rice Os11N3
promoter that contains the AvrXa7 EBE binding site (G). All three TALNs were successfully overproduced and purified from E. coli
cells (AvrXa7 TALNs, Supplementary Figure S2
). FN-AvrXa7 and AvrXa7-FN each were shown to bind specifically to double-stranded oligonucleotides containing the AvrXa7 EBE target site, but not to a slightly modified version of the binding site in an EMS assay (). Moreover, the purified AvrXa7-FN and FN-AvrXa7 TALNs exhibited cleavage activity near the expected EBE binding site under optimized reaction conditions in an in vitro
assay, the results of which were confirmed by DNA sequencing ( and ). Likewise, PthXo1-FN (alone and together with AvrXa7-FN) was shown to specifically cleave at its specific EBE DNA target site and produce the predicted sized DNA fragments (). Finally, expression of the chimeric FN-AvrXa7, AvrXa7-FN and PthXo1-FN TALNs in yeast stimulated HR between internal repeats of a disrupted and non-functional reporter gene (LacZ
) that contained appropriately paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sites (). These observations demonstrate the successful creation of functional TALNs and lead the way to future experimentation directed toward development of a technology for high-specificity gene knockout and HR in organisms that currently lack the ability to support either process in a practical manner for laboratory research.
FokI and its fusion proteins with zinc finger DNA-binding domains have been extensively studied. The endonuclease domain (FN) by itself has no specificity for cleavage, but cuts DNA at a set distance from the binding site specified by the FokI DNA-binding domain when the two domains are linked together (13–15
). In this sense, several types of FN based fusion proteins have been successfully created that combine new DNA sequence binding specificities with the FN cleavage activities, with ZFNs being the most familiar (6
). Study has shown that fusion of FN to ZF motif does not change the DNA-binding specificity of the ZF protein although it may cause slight decrease in binding affinity (41
). We chose the FokI cleavage domain to fuse with members of the TAL effector family and, as a proof of principle, demonstrated the feasibility and generality of creating a new class of rare-cutting, site-specific DNA nucleases with sequence specificities attributable to the TAL effectors. The DNA-binding features of TAL effectors make this group of proteins or their repetitive domains desirable as the key component of such chimeric endonucleases for a number of applications, including various sorts of genome editing. For example, the majority of naturally occurring TAL effector proteins contains a large number of repeat units and, correspondingly, recognizes lengthy DNA target sites (32
). These TAL EBE sites are comparable in length to, or longer than, target sites of rare-cutting meganucleases or homing nucleases (i.e. 14–40
bp) as well as binding sites for artificial zinc finger proteins assembled from multiple single fingers (i.e. 18 or 24
). All TAL effector proteins investigated thus far exhibit high sequence specificity to the EBEs of their target genes (32
). The known code of TAL effectors predicts an alignment of a single type of repeat unit to a single nucleotide species (A, G, C or T) based on the specific di-residues at positions 12 and 13 in the repeat unit. This modular nature of the TAL repeat domain for DNA-binding specificity suggests that techniques can be developed to produce an array of repeat units that can precisely recognize a unique, lengthy sequence of nucleotides in any given gene. If so, investigators will be able to create truly gene-specific TALNs for use in organisms with large genomes and lacking robust systems for HR.
Thus far, several TAL effectors have been found to function as transcription activators. Like many other transcription factors, TAL effectors may function as dimers to bind target DNA. However, to date, AvrBs3 is the only TAL effector shown to dimerize in vitro
and in the cytoplasm in vivo
before entry into nuclei of host cells (44
). The sequence specificity of known TAL effectors that bind DNA can be aligned to only one strand of the target site which is usually asymmetric (28
). Thus, it is not yet clear if most TAL effector proteins form dimers or multimers in the presence of target DNA (or in the absence of DNA). The results from the present yeast SSA assay imply that TALNs do not form homo- or hetero-dimer at a single TAL EBE site, or at least the dimerization of TAL subdomain does not facilitate the dimerization of FokI nuclease domains for effective double stranded DNA cleavage in yeast cells. More detailed structural studies of TAL effectors or TALNs likely will be needed to resolve this uncertainty, which may or may not negatively influence the ability to easily and successfully design sequence specific TALNs in the future.
It has been established that for efficient double strand cleavage of target DNA dimerization of FokI monomer nuclease domains is required (11
). Therefore, it is conceivable that TALNs need to dimerize for the efficient cleavage of DNA in solution where sufficient concentrations of purified proteins and substrates are present or in vivo
where TALN and target substrate are otherwise limited. This could be achieved through various mechanisms, three of which are presented below. In one model, one EBE-bound TALN might form a dimer with another bound or unbound TALN through an as yet uncharacterized dimerization motif of TAL effector. In such a case, the TAL subdomain-mediated dimerization could bring the two FokI nuclease domains in close proximity near the binding site and allow DNA cleavage. Alternatively, one TALN monomer could bind to one EBE target site and, similar to the model proposed for the native FokI or hybrid ZFNs in vitro
), dimerization of two DNA bound-TALNs through the well characterized dimerization motif in the FokI nuclease subdomain could occur in close proximity and, thereby, support DNA cleavage in trans
if sufficient concentrations of nucleases were present. Successful in vitro
cleavage of DNA carrying a single AvrXa7 EBE by the FN-AvrXa7, AvrXa7-FN and PthXo1-FN TALNs () is consistent with this model. In a third model in which two tandem, head-to-head or tail-to-tail EBE sites are present, TALNs could bind to each of the EBE sites. This would bring the two FN domains of the two TALNs into sufficiently close proximity to allow dimerization and DNA cleavage. Our yeast SSA data () is consistent with this latter model.
The function of native FokI is allosterically regulated through DNA and divalent metal binding. Without DNA binding and in the absence of divalent metal, FN is sequestered through tight interaction with the DNA recognition motifs of FokI and, thus, the FokI monomer maintains an idle state. Following binding of two FokI holoenzymes to the FokI recognition site and in the presence of metals, the two FokI nuclease domains are freed and can dimerize. This dimerization then allows double stranded DNA cleavage (14
). It is possible that the interaction between the FN subddomain and the TAL DNA-binding subdomain in hybrid TALN lacks such tight regulatory mechanism and, hence nuclease domains form dimers with less difficulty. That may explain why the apparent stringency of cleavage by the presently studied chimeric TALNs (and also ZFNs) is lower and leads to the non-specific cleavage observed in the presence of excess of TALNs (Supplementary Figure S3
). The structure (length and composition) of linker segment between the DNA-binding and -cleaving domains of FokI and its derived nucleases (i.e. ZFNs and, likely, TALNs) also dictate the enzymes’ cleavage pattern, for example, the distance of cleavage sites from the DNA-binding site (45–47
). The linker segment of native FokI is 15 aa (residues 373–387) long and allows the FN to extend and cleave the sense strand 9
bp and antisense strand 13
bp downstream of the binding site (12
). An 18 aa flexible linker of a ZFN accommodates effective cleavage of target spacer in a range of 6–18
bp with 8
bp as an optimum in Xenopus
oocytes as determined in a single-strand annealing reporter assay (45
). The latter study also reported that the dependence of efficient cleavage on spacer length in vitro
differed from that under in vivo
condition. Because the minimum-sized TAL effector fragment required for efficient DNA binding is unknown, the full-length AvrXa7 and PthXo1 were used to construct the TALNs in our study. Therefore, the N-terminal 288 aa in FN-AvrXa7 and the C-terminal 295 aa in AvrXa7-FN and PthXo1-FN function as long inter-domain linkers between FN and the repeat DNA-binding domain in TALNs. Such an extended inter-domain linker may allow significant ‘reach’ for the nuclease domain to cut at a moderate distance away from the ends of the EBE or allow greater flexibility for the nuclease domain to cut within a moderately wide zone as we observed in vitro
with our TALN enzyme assays and in vivo
with our yeast assays. Future investigations will be required to determine how various combinations of inter-domain sequence and length affects the cleavage efficiency of TALNs [e.g. tests of TALNs consisting of the FN connected directly to the TAL effector repeat domain or through linkers of various lengths and composition].
Other important questions are presently under active investigation. One such question is the nature of the DNA fragment with an apparent molecular size >10
kb when supercoiled plasmid DNA is mixed with a TALN (lanes 4 and 7 in B, lanes 4 and 6 in 3E). Such a band of DNA is not observed if the supercoiled plasmid is cleaved before mixing with the TALN (B and E). Our working hypothesis is that the uppermost, slow migrating DNA band may be a complex between plasmid DNA and the TALN protein that exists prior to DNA cleavage (by the TALN or a restriction enzyme), but not after cleavage. Future analyses of the component(s) of this DNA band (i.e. ethidium bromide stained band) should resolve this present enigma. Another issue is TALN stability. We have observed that all of the TALNs created for the present study have quite short half-lives (i.e. ~30–45
min). This currently imposes serious practical constraints on the degree of purity of recombinant TALNs that can be obtained and on the duration and extensiveness of biochemical analyses, including accurate measurements of TALN binding affinities to DNA. Further attempts to discover conditions that stabilize TALNs overproduced in E. coli
will be important undertakings along with endeavors to determine if such instability does or does not exist in vivo
in various cell types.
The work presented here unambiguously demonstrates several points: the ability to fuse TAL effectors with other proteins to create functional chimeric proteins; the specificity of DNA binding by TAL effectors when fused with a non-specific nuclease domain; and the specific cleavage of DNA target sites by engineered TALNs both in vitro
and in vivo
. The newly emerging TALN-based approach could be an attractive alternative to the still improving ZFN-based or meganuclease-based (48
) genomic tools for a wide variety of possible applications including targeted genome editing. However, a few basic questions remain unanswered regarding the feasibility of using TALNs for genome modification. First, can novel TALN DNA-binding domains with the requisite specificity and affinity be synthesized based on the actual DNA target sequences? Although arbitrarily assembled, TAL effectors were able to activate promoters containing sequence elements synthesized based on the ‘code’ (29
), this capability is not yet demonstrated for a TALN fusion protein. Second, can two different TALNs work coordinately at preselected adjacent target sites? The reaction involving cleavage of a dual asymmetric PthXo1 EBE/AvrXa7 EBE site with a mixture of PthXo1-FN and AvrXa7-FN (E, lane 7) hints this may be possible. That is, the two TALNs together produced somewhat better, albeit still incomplete, plasmid cleavage than when each TALN was used independently at five times the concentration employed in the dual cutting reaction—and with less non-specific plasmid DNA cleavage (compare E, lanes 5 and 7 with lane 8). Third, will the DNA recognition and cleavage by the TALNs occur in a chromosomal context in living cells? Data from the yeast experiment described in this paper () provide strong initial evidence that a TALN or sets of TALNs can successfully find, bind and cleave an EBE target site within yeast chromatin. Addressing these issues will assist in achieving the goal of generating a tool box of TALNs for targeted genome editing based on the DNA-binding specificity of custom-designed, synthetic TAL repeat domains and the DNA-cleavage function of FokI or other nucleases.