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. 2015 Aug;25(8):1158-69.
doi: 10.1101/gr.179044.114. Epub 2015 May 29.

Genome-wide Specificity of DNA Binding, Gene Regulation, and Chromatin Remodeling by TALE- And CRISPR/Cas9-based Transcriptional Activators

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Free PMC article

Genome-wide Specificity of DNA Binding, Gene Regulation, and Chromatin Remodeling by TALE- And CRISPR/Cas9-based Transcriptional Activators

Lauren R Polstein et al. Genome Res. .
Free PMC article

Abstract

Genome engineering technologies based on the CRISPR/Cas9 and TALE systems are enabling new approaches in science and biotechnology. However, the specificity of these tools in complex genomes and the role of chromatin structure in determining DNA binding are not well understood. We analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators in human cells using ChIP-seq to assess DNA-binding specificity and RNA-seq to measure the specificity of perturbing the transcriptome. Additionally, DNase-seq was used to assess genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these transcription factors are highly specific in both DNA binding and gene regulation and are able to open targeted regions of closed chromatin independent of gene activation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function.

Figures

Figure 1.
Figure 1.
Targeted activation of the human IL1RN and HBG1/2 genes by TALE-VP64 and dCas9-VP64 transcription factors. (A) Four TALEs (blue) and four gRNAs (orange), each labeled A through D, were designed to target the IL1RN and HBG1/2 promoters within the ∼200 bp upstream of the transcriptional start site (TSS; green). The position of each TALE and gRNA is shown to scale. (B) Single expression plasmids or combinations of two, three, or four expression plasmids for the TALE-VP64s or gRNAs, along with dCas9-VP64, targeted to each gene were transfected into HEK293T cells. Expression of the target gene was assessed by qRT-PCR. Robust gene activation was observed only in response to the combination of TALE-VP64s or gRNAs with dCas9-VP64. (C) The VP64 activation domain is essential for target gene induction. HEK293T cells were transfected with the combination of expression plasmids for the four TALEs with and without the VP64 domain, or four gRNAs either alone or with dCas9 or dCas9-VP64. Only samples transfected with TALE-VP64s or gRNAs with dCas9-VP64 showed changes in target gene expression. Gene expression is normalized to GAPDH levels and shown as fold-increase relative to control cells transfected with an empty expression plasmid (mean ± SEM, n = 4 independent transfections across two experiments, different letters indicate P < 0.0001 by Tukey's test after log transformation).
Figure 2.
Figure 2.
Genome-wide specificity of TALE-VP64 and dCas9-VP64-mediated gene activation. RNA-seq was performed on samples co-transfected with a set of four TALE-VP64 expression plasmids targeting either IL1RN (A) or HBG1/2 (C). In each case, the only genome-wide significant changes (false discovery rate <5%) in gene expression between the treatments and an empty plasmid-transfected control were increases in the expression of IL1RN or HBG1/2, respectively. (B,D) Comparison of RNA-seq measurements of gene expression after activating expression using TALE-VP64 (x-axis) or dCas9-VP64 (y-axis). (B) When targeting IL1RN, the TALE-VP64-mediated activation was slightly stronger. (D) When targeting HBG1/2, TALE-VP64 had a substantially stronger effect on expression. RNA-seq for dCas9-VP64 samples was published previously (Perez-Pinera et al. 2013a).
Figure 3.
Figure 3.
Genome-wide specificity of dCas9-VP64 and TALE-VP64 DNA-binding. ChIP-seq was used to map the genomic locations of dCas9-VP64 targeted to the IL1RN promoter (A), TALE-VP64 targeted to the IL1RN promoter (B), dCas9-VP64 targeted to the HBG1/2 promoters (C), or TALE-VP64 targeted to the HBG1/2 promoters (D). In each plot, points are binding sites that are reproducible in at least two of three replicates. The x-axes are the mean ChIP-seq signal, and the y-axes are the fold-change in signal in samples transfected with dCas9-VP64 or TALE-VP64 transcription factors compared to controls. Red points represent binding sites with a statistically significant increase in signal strength according to analysis with DESeq (false discovery rate <0.1%). (E) As an example of the genome-wide binding specificity dCas9-VP64 and TALE-VP64 targeting, ChIP-seq signal from experiments targeting HBG1/2 was plotted across Chromosome 11. ChIP-seq signals found in both experimental conditions and in the control condition are also found in the ENCODE blacklist, indicating that they are technical artifacts of ChIP-seq and not binding events. Meanwhile, strong ChIP-seq signal was found at the HBG1/2 promoters in the dCas9-VP64 and TALE-VP64 conditions but not in the control condition. (F) The dCas9-VP64 and TALE-VP64 ChIP-seq peaks localize to the intended HBG1/2 promoters.
Figure 4.
Figure 4.
Characterization of dCas9-VP64 and TALE-VP64 off-target binding sites. (A) De novo motif detection was used to identify gRNAs and TALEs responsible for identified off-target binding sites. For dCas9-VP64 targeted with gRNAs, motifs matching two of the IL1RN gRNAs and one of the HBG1/2 gRNAs were identified in the respective binding sites identified with ChIP-seq. For TALE-VP64, no motifs matching the IL1RN TALEs were identified, and one motif matching a HBG1/2 TALE-VP64 was identified. (B) For each off-target binding site identified by ChIP-seq, we performed an unbiased search for sequences that resemble the intended target sequences of each of the gRNAs or TALE identified in A. For this analysis, we considered every possible binding sequence in the called ChIP-seq peak. For dCas9-VP64, we required each possible binding sequence to be followed by the “NGG” PAM sequence. For TALE-VP64, every position in the called binding site was used. Next, for each of the three gRNAs or TALEs identified in A, we aligned the intended target sequence to that of every possible binding sequence, and the sequence with the most matching nucleotides in each binding site was retained. DNA sequence similarity to the target sequence at the matched sites was then plotted as a function of the position in the target sequences. For the three gRNAs investigated, a statistically significant trend toward more similarity at the 3′ end of the gRNA sequence was identified, indicating that the 3′ end of the gRNA is more influential in guiding dCas9-VP64 binding. In contrast, the weak 3′ trend observed for TALE D binding is likely an artifact of low sequence complexity in the 3′ end of the target sequence.
Figure 5.
Figure 5.
Chromatin accessibility changes induced by both TALEs and dCas9. HEK293T cells were transfected with expression plasmids for TALEs ± VP64 and gRNAs with dCas9 ± VP64 targeted to either the HBG1/2 promoter or the IL1RN promoter. (A) Representative DNase-seq data surrounding each promoter (highlighted in box) show increased chromatin accessibility at the promoter to which the TALEs and dCas9 are targeted, but not at the other promoter. (B) Normalized DNase-seq cut counts within a 300-bp window surrounding each promoter are shown (mean ± SEM, n = 4–6) (Supplemental Table 32). P-values are shown compared to the control sample (Tukey's test).
Figure 6.
Figure 6.
Global characterization of changes to chromatin accessibility. (A) Scatter plot of DNase-seq data comparing samples treated with TALEs, with and without VP64, targeted to IL1RN versus HBG1/2. Each dot represents a DNase I hypersensitive site analyzed by DESeq. IL1RN and HBG1/2 display the expected opposite differences in chromatin accessibility. Nominal P-values for each target site are indicated. (B) Similar scatter plot as A, but for DNase-seq data from IL1RN-targeted dCas9 ± VP64 versus HBG1/2-targeted dCas9 ± VP64. The individual comparisons of all eight treatments compared to control are presented in Supplemental Figures 5, 6; and the top 100 differential DHS sites for each treatment are provided in Supplemental Tables 16–23. (CF) DNase-seq signal for target (red circles) and off-target ChIP-seq sites (black circles). For each off-target ChIP-seq site, normalized DNase-seq signal from IL1RN-targeted TALE-VP64 (C), IL1RN-targeted dCas9-VP64 (D), HBG1/2-targeted TALE-VP64s (E), and HBG1/2-targeted dCas9-VP64 (F) was compared to normalized DNase-seq signal from control cells transfected with empty plasmid.

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