Efficient construction of sequence-specific TAL effectors for modulating mammalian transcription

Abstract

The ability to direct functional domains to specific DNA sequences is a long sought-after goal for studying and engineering biological processes. Transcription activator like effectors (TALEs) from Xanthomonas sp. present a promising platform for designing sequence-specific DNA binding proteins. Here we describe a robust and rapid method for overcoming the difficulty of constructing TALE repeat domains. We synthesized 17 designer TALEs (dTALEs) that are customized to recognize specific DNA binding sites, and demonstrate that dTALEs can specifically modulate transcription of endogenous genes (Sox2 and Klf4) from the native genome in human cells. dTALEs provide a designable DNA targeting platform for the interrogation and engineering of biological systems.

Crystal structure of a TALE protein reveals an extended N-terminal DNA binding region

“…A truncated dTale2 protein (230-610), which is similar to the DNA-bound dHax3 with a premature NTR [16], bound to specified DNA2 with a lower affinity (7.81 µM) ( Figure 1J), indicating that the intact NTR is crucial for the DNA binding ability of dTale2 (148-610), consistent with previous reports that suggest that a fragment encompassing more than 100 residues of the NTR is essential for the full activities of customized TALE fusion proteins [11][12][13][14]. The dTale2 variant protein (148-766) with an extended C-terminal region, bound to specified DNA2 with a comparable affinity (0.70 µM) to dTale2 (148-610) Haishan Figure S10A and S10B), suggesting that dTale2 (148-610) was sufficient to bind to the target DNA.…”

“…As shown by the ITC assay, the single mutation (K169A, W232A or R236A) had little effect on the DNA binding ability of dTale2 (148-610), whereas the multiple-mutations of the positively charged amino acids to alanines (K262A/K265A/R266A), (K171A/K262A/ K265A/R266A), (R173A/K262A/K265A/R266A) and (K230A/Q231A/K262A/K265A/R266A) greatly impaired the DNA binding activity of dTale2 (148-610) (Supplementary information, Table S3), implying that these basic amino acids are vital for DNA binding and play synergetic roles in interacting with DNA. Coupled with the previous data from TALEs' applications in vivo [11][12][13][14], it is suggested that the NTR serves as the indispensable "nucleation site" for the TALE-DNA binding both in vitro and in vivo.…”

Specific inhibition of diverse pathogens in human cells by synthetic microRNA-like oligonucleotides inferred from RNAi screens

“…At least some of these off-target effects are presumably mediated by the cellular microRNA-processing machinery, which mistakes transfected siRNA oligos for endogenous microRNAs, loading them onto the RNA-induced silencing complex and scanning for mRNAs with suitable binding sites. Consistent with this hypothesis, it has been observed that sequencedependent off-target effects of siRNAs are primarily controlled and initiated by the "seed" region of their sequence (nucleotide positions [2][3][4][5][6][7][8], similar to what is the case for microRNAs (6,19,20). Matches to any given seed sequence typically occur in several hundred different human transcripts, suggesting that each off-target event can potentially perturb tens or hundreds of genes simultaneously.…”

“…In contrast, when working with human cells, the technical possibilities for gene perturbations are much more limited. Although promising technologies for targeted genome editing in human cells have been introduced recently (2)(3)(4)(5), these are at present too cumbersome for routine, genome-wide screening.…”

Genome-Editing Technologies: Principles and Applications

“…Over the course of the next 15 years, zinc-finger-based transcriptional modulators were expanded and featured several other types of effector domains (Beerli and Barbas 2002), including, for example, the Dnmt3a methyltransferase domain (Rivenbark et al 2012;Siddique et al 2013) and the ten-eleven translocation methylcytosine dioxygenase 1 (TET1) , which can modulate transcription via targeted methylation or demethylation, respectively. As a natural extension of zinc-finger transcription factors, and further drawing on the parallels with zinc-finger proteins, TALE transcription factors have also emerged as an especially effective platform for achieving targeted transcriptional modulation Zhang et al 2011). Effector domains are generally fused to the carboxyl terminus of the synthetic TALE array and, contrary to the longer sequence typically required for efficient modulation by zinc-finger transcription factors, TALEs have been reported to regulate gene expression with as few as 10.5 repeats (Boch et al 2009).…”