Engineering synthetic TAL effectors with orthogonal target sites

Abstract

The ability to engineer biological circuits that process and respond to complex cellular signals has the potential to impact many areas of biology and medicine. Transcriptional activator-like effectors (TALEs) have emerged as an attractive component for engineering these circuits, as TALEs can be designed de novo to target a given DNA sequence. Currently, however, the use of TALEs is limited by degeneracy in the site-specific manner by which they recognize DNA. Here, we propose an algorithm to computationally address this problem. We apply our algorithm to design 180 TALEs targeting 20 bp cognate binding sites that are at least 3 nt mismatches away from all 20 bp sequences in putative 2 kb human promoter regions. We generated eight of these synthetic TALE activators and showed that each is able to activate transcription from a targeted reporter. Importantly, we show that these proteins do not activate synthetic reporters containing mismatches similar to those present in the genome nor a set of endogenous genes predicted to be the most likely targets in vivo. Finally, we generated and characterized TALE repressors comprised of our orthogonal DNA binding domains and further combined them with shRNAs to accomplish near complete repression of target gene expression.

Figure 1.

Orthogonal TALEs as ideal regulatory components for insulated synthetic gene circuits. (A) Non-orthogonal TALEs designed to bind and regulate gene expression of a synthetic gene circuit may also bind to cognate and off-target (containing mismatches) binding sequences in the endogenous promoter regions in the genomic DNA. (B) Orthogonal TALEs bind and regulate gene expression of a synthetic gene circuit and have no predicted binding sites in the endogenous promoter regions.

Figure 2.

TALE protein architecture and DNA binding specificities. (A) Schematic of a representative TALE protein with 18.5 repeat variable di-residue (RVD) domains. Each RVD domain is composed of 34 amino acids and differs only in the variable amino acids highlighted in red. The C-terminal RVD domain is a 15 amino acid half repeat domain. The two endogenous NLS domains and the endogenous activation domain (AD) present in naturally occurring TALEs were replaced with SV40 NLSs and the VP64 activation domain, respectively. (B) The amino acid sequences and the preferred target nucleotides of RVD domains NI, HD, NG from AvrBs3 and RVD domain NK from pthA2.

Figure 3.

Flowchart enumerating the steps used in our algorithm to compute orthogonal 20-mers. Steps 1 and 2 describe the process used to reduce the set of genomic 20-mers. Steps 2 and 3 describe the process of obtaining 20-mers orthogonal to the genomic set. Steps 2 and 3 of the algorithm can be iterated until the desired number of orthogonal sequences has been computed. Finally, the resulting sets of TALEs are checked for mutual orthogonality to avoid cross-interference within the synthetic circuits.

Figure 4.

(A) Schematic of TALE expression constructs. Each TALE coding region was cloned into a mammalian expression vector downstream of the CMV promoter. All synthetic TALEs were also tagged with an self-cleaving t2A:mCherry fluorescent protein as a transfection control. (B) Schematic of TALE reporter constructs. Reporter constructs were generated by cloning a 20 bp TALE target sequence upstream of a minimal TATA box separated by a 78 bp spacer region. Binding of a TALE activator to the 20 bp target sequence drives expression of two tandem copies of NLS-tagged CFP cloned downstream of the TALE-responsive promoter as an output for TALE functionality.

Figure 5.

Functional characterization of TALE activators. (A) Fluorescence microscopy images of TALE-induced CFP reporter expression. Each column of the 8 × 8 matrix represents U2-OS cells co-transfected with a synthetic TALE and reporter constructs for each 20-mer binding site (BS). The CFP signal is only visible along the diagonal of the matrix, indicating that the TALEs described here function in a mutually orthogonal manner. (B) Bar graphs representing mutually orthogonal TALE activity as determined by flow cytometry. The fold induction of CFP expression, as calculated relative to an off-target control TALE, displays values ranging from approximately 10-fold to 100-fold for cognate target sites and demonstrates the functionality and mutual orthogonality of these TALEs.

Figure 6.

Effect of binding site mutations on TALE-mediated transcriptional activation. (A) TALE8 activity in the presence of an increasing number of uniformly distributed binding site mismatches. BS-8 is the corresponding binding site for TALE8 with additional binding sites tested at a hamming distance (HD) of 1–7 bp from BS-8 (HD-1–HD-7). The ability of TALE8 to activate CFP expression from each binding site reporter was measured by flow cytometry relative to TALE5 as an off-target control. The presence of two or more mismatches in the binding site significantly decreases the ability of TALE8 to activate gene expression, with binding sites at a hamming distance of more than 3 bp displaying no reporter activity. (B) Effect of binding site mismatch position on TALE activation. The ability of TALE8 to activate gene expression from binding sites with a hamming distance of 3 bp was tested with the position of the mismatches either uniformly distributed, HD-3, localized to the 5′-end of the binding site, HD-3(5′), to the middle of the binding site, HD-3(M) or to the end of the binding site, HD-3(3′). (C) Tested DNA binding sequences. Underlined nucleotides represent mismatches with respect to BS-8.

Figure 7.

Characterization of TALE-mediated off-target endogenous gene activation in vivo. Fold change in mRNA levels of potential target genes following TALE expression. mRNA levels of the most likely target genes of TALE5 and TALE8 were measured by qPCR 48 h post-transfection with the corresponding TALE construct and plotted as fold change over mock-transfected cells. TALE-OSGIN2 and TALE-ZC3H10 are the positive control TALEs predicted to activate the two closest off-target genes of TALE5 and TALE8 respectively. (A) A 4.8-fold induction of nearest target gene OSGIN2 by the positive control TALE-OSGIN2, and no significant change in mRNA levels of OSGIN2 and the other four nearest target genes of TALE5 is observed in response to TALE5. The 10× higher concentration (250 ng) of TALE5 also shows no significant induction in mRNA levels of its off-target gene OSGIN2. (B) The positive control TALE-ZC3H10 leads to a modest but significant induction of nearest target gene (ZC3H10) of TALE8. There is no significant change in mRNA levels of the four nearest target genes of TALE8 in response to TALE8 expression. Asterisk indicates P < 0.03.

Figure 8.

Schematics and characterization of TALE repressor-shRNA constructs. (A) The VP64 activation domain of the TALE activators was replaced with the KRAB repression domain and the resulting TALE repressor coding region was cloned into a mammalian expression vector together with a self-cleaving DsRed:t2A fluorescent protein. Synthetic shRNAs were expressed from an intron in the DsRed gene. (B) Reporter constructs were generated by cloning a 20 bp TALE target sequence into the TSS of the CMV promoter. On binding its recognition site in the promoter, the TALE represses the constitutive expression of the downstream CFP protein. The reporter construct also contains a single copy of the cognate shRNA recognition sequence in the 3′-UTR, which when recognized by the target shRNA leads to degradation of the CFP transcript. (C) TALE repressors, TALE5R and TALE8R were combined with shRNAs, FF4 and FF6, to repress CFP expression from reporter constructs carrying cognate TALE and shRNA recognition sites. Repressions ranged 6× in the case of shRNA alone to ∼4800× in the case of shRNA + TALE repressor combination.