Targeted genetic engineering of human pluripotent cells is a prerequisite for exploiting their full potential. Such genetic manipulations can be achieved using site-specific nucleases. Here, we engineered Transcription Activation-Like Effector Nucleases (TALENs) for five distinct genomic loci. At all loci tested we obtained hESC and iPSC single-cell-derived clones carrying transgenic cassettes solely at the TALEN-specified location. Thus, TALENs mediate site-specific genome modifications in human pluripotent cells with comparable efficiency and precision as zinc finger nucleases (ZFNs).

Clathrin-mediated endocytosis (CME) is the best-studied pathway by which cells selectively internalize molecules from the plasma membrane and surrounding environment. Previous live-cell imaging studies using ectopically overexpressed fluorescent fusions of endocytic proteins indicated that mammalian CME is a highly dynamic but inefficient and heterogeneous process. In contrast, studies of endocytosis in budding yeast using fluorescent protein fusions expressed at physiological levels from native genomic loci have revealed a process that is very regular and efficient. To analyse endocytic dynamics in mammalian cells in which endogenous protein stoichiometry is preserved, we targeted zinc finger nucleases (ZFNs) to the clathrin light chain A and dynamin-2 genomic loci and generated cell lines expressing fluorescent protein fusions from each locus. The genome-edited cells exhibited enhanced endocytic function, dynamics and efficiency when compared with previously studied cells, indicating that CME is highly sensitive to the levels of its protein components. Our study establishes that ZFN-mediated genome editing is a robust tool for expressing protein fusions at endogenous levels to faithfully report subcellular localization and dynamics.

RNA binding proteins of the conserved CUGBP1, Elav-like factor (CELF) family contribute to heart and skeletal muscle development and are implicated in myotonic dystrophy (DM). To understand their genome-wide functions, we analyzed the transcriptome dynamics following induction of CELF1 or CELF2 in adult mouse heart and of CELF1 in muscle by RNA-seq, complemented by crosslinking/immunoprecipitation-sequencing (CLIP-seq) analysis of mouse cells and tissues to distinguish direct from indirect regulatory targets. We identified hundreds of mRNAs bound in their 3 ′ UTRs by both CELF1 and the developmentally induced MBNL1 protein, a threefold greater overlap in target messages than expected, including messages involved in development and cell differentiation. The extent of 3 ′ UTR binding by CELF1 and MBNL1 predicted the degree of mRNA repression or stabilization, respectively, following CELF1 induction. However, CELF1's RNA binding specificity in vitro was not detectably altered by coincubation with recombinant MBNL1. These findings support a model in which CELF and MBNL proteins bind independently to mRNAs but functionally compete to specify down-regulation or localization/stabilization, respectively, of hundreds of mRNA targets. Expression of many alternative 3 ′ UTR isoforms was altered following CELF1 induction, with 3 ′ UTR binding associated with down-regulation of isoforms and genes. The splicing of hundreds of alternative exons was oppositely regulated by these proteins, confirming an additional layer of regulatory antagonism previously observed in a handful of cases. The regulatory relationships between CELFs and MBNLs in control of both mRNA abundance and splicing appear to have evolved to enhance developmental transitions in major classes of heart and muscle genes.[Supplemental material is available for this article.] CELF RNA binding proteins (RBPs) play roles in early embryonic development, heart, and skeletal muscle functions. They are also thought to contribute to DM pathogenesis (Timchenko et al. 1996) and have been suggested to contribute to other diseases (Ladd 2013). The six family members present in mammals can be divided into two subfamilies: CELF1-2, which are expressed most highly in heart, skeletal muscle, and brain, and CELF3-6, which exhibit more restricted expression (Dasgupta and Ladd 2012). The CELF proteins contain two N-terminal RNA recognition motifs (RRMs) and one C-terminal RRM, with which they bind GU-rich RNAs, and a linker region termed the "divergent domain" that separates RRM2 and RRM3 and is involved in the regulation of alternative pre-mRNA splicing and mRNA decay (Han and Cooper 2005;.During normal development, CELF1 and CELF2 proteins are highly expressed in early embryonic stages and are then down-regulated more than 10-fold in skeletal muscle (Ladd et al. 2005) and the heart (Kalsotra et al. 2008) during post-natal development, remaining at low levels in adult tissues. This developmental downregulation occurs through multiple mechanisms, including repression by microRNAs (m...

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The frog Xenopus , an important research organism in cell and developmental biology, currently lacks tools for targeted mutagenesis. Here, we address this problem by genome editing with zinc-finger nucleases (ZFNs). ZFNs directed against an eGFP transgene in Xenopus tropicalis induced mutations consistent with nonhomologous end joining at the target site, resulting in mosaic loss of the fluorescence phenotype at high frequencies. ZFNs directed against the noggin gene produced tadpoles and adult animals carrying up to 47% disrupted alleles, and founder animals yielded progeny carrying insertions and deletions in the noggin gene with no indication of off-target effects. Furthermore, functional tests demonstrated an allelic series of activity between three germ-line mutant alleles. Because ZFNs can be designed against any locus, our data provide a generally applicable protocol for gene disruption in Xenopus .