Mutation rate mediated by TALEN pairs in transfected 293T cells.

<p>Mutation rate mediated by TALEN pairs in transfected 293T cells.</p

Using two TALEN pairs to delete the entire human miR-155 hairpin sequence.

<p>(A) TALEN pairs targeting miR-155 were designed and constructed (called TALEN C). The upper panel shows a schematic of the TALEN A pair binding sites. The lower panel shows the sequence alignments comparing Wt and TALEN C mutated miR-155. The left and right TALEN binding sites are highlighted in yellow and the miR-155 seed region is boxed in red. (B) Schematic of the binding sites of two TALEN pairs (TALEN A and TALEN C) targeting miR-155. (C–D) Both TALEN A and TALEN C pairs were transfected into 293T cells. The miR-155 locus was amplified by PCR and subjected to TOPO cloning and sequencing. (C) Electrophoresis gel analysis showing deletions in the miR-155 locus. The arrows on the right indicate the two expected PCR products with or without large deletions. (D) Sequence alignments between a Wt clone and two TOPO clones with large deletions. The left and right TALEN binding sites for both TALEN A and TALEN C are highlighted in yellow and the miR-155 hairpin sequence is boxed in red.</p

The miR-155* targeting TALEN pair causes mutations in the miR-155* sequence.

<p>293T cells were transfected with or without plasmids encoding the TALEN pair designed to target the miR-155* region. A GFP expressing plasmid was co-transfected. (A) 48 hours later, GFP+ cells were sorted by FACS and subjected to gDNA extraction. (B–F) The miR-155* targeted region was amplified by PCR and subjected to an HRMA analysis (B–D) or TOPO cloning and sequencing (E,F). (B) Schematic of the HRMA approach. A small region of the genome that includes different lengths of DNA deletions is amplified by PCR. Upon annealing, different types of homoduplex and heteroduplex dsDNA molecules are produced with different melting temperatures. (C) HRMA of the mir-155* PCR amplicons generated using gDNA from Wt (mock transfected 293t cells), Unsorted (293t cells with the TALEN pair transfection), Sorted GFP+ and sorted GFP-(293t cells with the TALEN pair and a GFP plasmid co-transfection followed by FACS sorting). (D) The results of the HRMA analysis are also shown as fluorescence difference plots using the normalized data. The Wt sample is used as the baseline. (E) PCR products from C were cloned into a TOPO vector and the length of the individual DNA fragment was assessed by gel electrophoresis. (F) Sequencing results of TOPO clones from E are shown. They are aligned with the wild-type miR-155* sequence. The left and right TALEN binding sites are highlighted in yellow and the miR-155* region is boxed in red.</p

Using TALENs to target the seeds of human miR-146a and miR-125b1.

<p>TALEN pairs targeting the indicated miRNAs were designed and constructed. After the TALEN pairs were transfected into 293T cells, methods were performed (as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063074#pone-0063074-g002" target="_blank">Figure 2</a>) to detect mutations in the targeted regions. (A, B) The upper panel shows the schematics of the TALEN pair binding sites adjacent to (A) miR-146a and (B) miR-125b1. The lower panel shows the sequence alignments between the Wt and mutated miRNA genes. The left and right TALEN binding sites are highlighted in yellow and the miRNA seed regions are boxed in red.</p

Schematic of the miR-155/miR-155* genomic locus and the location of the TALEN pair engineered to target the miR-155* region.

<p>(A) Schematic of the miR-155/miR-155* genomic locus. The three BIC exons are shown in yellow and the miR-155 hairpin is in red. (B) Schematic of the miR-155 hairpin structure. The miR-155 arms are shown in black, while the mature miR-155 and miR-155* sequences are in dark grey. Blue and green boxes represent the binding sites of the TALEN pair designed to target the miR-155* region, and hexagons represent the heterodimerized FokI enzyme positioned over the spacer sequence. (C) The two expression plasmids containing the TALEN pair along with the FokI nuclease domain, and the TALEN-RVD sequences corresponding to each targeted DNA sequence are shown. The details of pCS2TAL3-DDD and pCS2TAL3-RRR expression vectors are described in the Materials and Methods section. NN, HD, NG and NI represent the RVD regions of each repeat sequence that bind to nucleotide G, C, T and A, respectively. The left and right TALEN binding sequences are shown in red and purple, respectively, and the spacer region is in blue.</p

The miR-155* targeting TALEN pair causes both bi-allelic and mono-allelic mutations in human cells.

<p>(A) Schematic of the experimental design. 293T cells were transfected with the TALEN pair targeting the miR-155* region along with a GFP plasmid. 48 hours later, GFP+ cells were sorted by FACS and subjected to single cell cloning. After individual cell clones were expanded, gDNA was extracted from the cell clones. The miR-155* TALEN pair-targeted region was amplified by PCR and subjected to TOPO cloning and sequencing. (B,C) Representative cell clones showing bi-allelic mutations (B) or mono-allelic mutations (C). In the sequence alignment graph, the left and right TALEN binding sites are highlighted in yellow and the miR-155* region is in the red box.</p

Targeting Human MicroRNA Genes Using Engineered Tal-Effector Nucleases (TALENs)

<div><p>MicroRNAs (miRNAs) have quickly emerged as important regulators of mammalian physiology owing to their precise control over the expression of critical protein coding genes. Despite significant progress in our understanding of how miRNAs function in mice, there remains a fundamental need to be able to target and edit miRNA genes in the human genome. Here, we report a novel approach to disrupting human miRNA genes <i>ex vivo</i> using engineered TAL-effector (TALE) proteins to function as nucleases (TALENs) that specifically target and disrupt human miRNA genes. We demonstrate that functional TALEN pairs can be designed to enable disruption of miRNA seed regions, or removal of entire hairpin sequences, and use this approach to successfully target several physiologically relevant human miRNAs including miR-155*, miR-155, miR-146a and miR-125b. This technology will allow for a substantially improved capacity to study the regulation and function of miRNAs in human cells, and could be developed into a strategic means by which miRNAs can be targeted therapeutically during human disease.</p></div

Anti-correlation functional profiling identifies relevant miRNA-target interactions, including miR-150 repression of p53, that regulate MV4-11 cell line growth.

<p>(A) Heat map indicating representative oncogenes whose loss leads to decreased cell growth according to Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and functionally anti-correlated miRNAs that are predicted to target each oncogene. Grey boxes indicate that the miRNA is not predicted to bind the 3’UTR of the oncogene (NT = Not targeted). (B) Heat map indicating representative TSGs whose loss lead to increased cell growth according to our Log2 Fold Change values from lentiCRISPRv2 library screen (first column), and miRNAs predicted to target each TSG whose growth anti-correlated in library. Grey boxes indicates that the miRNA is not predicted to bind the 3’UTR of TSG (NT = Not targeted). (C) Schematic showing miR-150 targeting of the p53 3’UTR. (D) Schematic of the miR-150 hairpin sequence as annotated in miRBase and sgRNA design of the miR-150-targeting lentiCRISPRv2 construct (150-CR1). (E) Expression level of miR-150 in MV4-11 cells infected with EV control or 150-CR1 lentiCRISPRv2 constructs determined by qPCR. Expression normalized to 5s. (F) Western blot of p53 in p53-CR1, 150-CR1, and EV control infected MV4-11 cell lines with actin serving as load control. (G) Competitive growth curve of EV (GFP+), p53-CR1 (GFP+), or 150-CR1 (GFP+) infected MV4-11 cells mixed ~1:1 with WT MV4-11 cells at time point 0. Y-axis = (%GFP+ cells at indicated time point)/(%GFP+ cells initial). (A, B) Only expressed protein-coding genes and microRNAs with p-values <0.05 were analyzed. Data represented as mean +/- SEM. P-values as indicated: *≤0.05, **≤0.01, ***≤0.001, and ns p>0.05. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153689#pone.0153689.s004" target="_blank">S3 Table</a>.</p

Genome-Wide CRISPR-Cas9 Screen Identifies MicroRNAs That Regulate Myeloid Leukemia Cell Growth

<div><p>Mammalian microRNA expression is dysregulated in human cancer. However, the functional relevance of many microRNAs in the context of tumor biology remains unclear. Using CRISPR-Cas9 technology, we performed a global loss-of-function screen to simultaneously test the functions of individual microRNAs and protein-coding genes during the growth of a myeloid leukemia cell line. This approach identified evolutionarily conserved human microRNAs that suppress or promote cell growth, revealing that microRNAs are extensively integrated into the molecular networks that control tumor cell physiology. miR-155 was identified as a top microRNA candidate promoting cellular fitness, which we confirmed with two distinct miR-155-targeting CRISPR-Cas9 lentiviral constructs. Further, we performed anti-correlation functional profiling to predict relevant microRNA-tumor suppressor gene or microRNA-oncogene interactions in these cells. This analysis identified miR-150 targeting of p53, a connection that was experimentally validated. Taken together, our study describes a powerful genetic approach by which the function of individual microRNAs can be assessed on a global level, and its use will rapidly advance our understanding of how microRNAs contribute to human disease.</p></div