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Polymorphic HLAs form the primary immune barrier to cell therapy. In addition, innate immune surveillance impacts cell engraftment, yet a strategy to control both, adaptive and innate immunity, is lacking. Here we employed multiplex genome editing to specifically ablate the expression of the highly polymorphic HLA-A/-B/-C and HLA class II in human pluripotent stem cells. Furthermore, to prevent innate immune rejection and further suppress adaptive immune responses, we expressed the immunomodulatory factors PD-L1, HLA-G, and the macrophage “don’t-eat me” signal CD47 from the AAVS1 safe harbor locus. Utilizing in vitro and in vivo immunoassays, we found that T cell responses were blunted. Moreover, NK cell killing and macrophage engulfment of our engineered cells were minimal. Our results describe an approach that effectively targets adaptive as well as innate immune responses and may therefore enable cell therapy on a broader scale.

Although human genetic studies have implicated many susceptible genes associated with plasma lipid levels, their physiological and molecular functions are not fully characterized. Here we demonstrate that orphan G protein-coupled receptor 146 (GPR146) promotes activity of hepatic sterol regulatory element binding protein 2 (SREBP2) through activation of the extracellular signal-regulated kinase (ERK) signaling pathway, thereby regulating hepatic very low-density lipoprotein (VLDL) secretion, and subsequently circulating low-density lipoprotein cholesterol (LDL-C) and triglycerides (TG) levels. Remarkably, GPR146 deficiency reduces plasma cholesterol levels substantially in both wild-type and LDL receptor (LDLR)-deficient mice. Finally, aortic atherosclerotic lesions are reduced by 90% and 70%, respectively, in male and female LDLR-deficient mice upon GPR146 depletion. Taken together, these findings outline a regulatory role for the GPR146/ERK axis in systemic cholesterol metabolism and suggest that GPR146 inhibition could be an effective strategy to reduce plasma cholesterol levels and atherosclerosis.

The use of human pluripotent stem cells for in vitro disease modelling and clinical applications requires protocols that convert these cells into relevant adult cell types. Here, we report the rapid and efficient differentiation of human pluripotent stem cells into vascular endothelial and smooth muscle cells. We found that GSK3 inhibition and BMP4 treatment rapidly committed pluripotent cells to a mesodermal fate and subsequent exposure to VEGF-A or PDGF-BB resulted in the differentiation of either endothelial or vascular smooth muscle cells, respectively. Both protocols produced mature cells with efficiencies exceeding 80% within six days. On purification to 99% via surface markers, endothelial cells maintained their identity, as assessed by marker gene expression, and showed relevant in vitro and in vivo functionality. Global transcriptional and metabolomic analyses confirmed that the cells closely resembled their in vivo counterparts. Our results suggest that these cells could be used to faithfully model human disease.

Summary: Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease—dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease. Highlights: ► A system for efficient and rapid genome editing with TALENs ► Generation of isogenic human cellular models of disease ► Identification of disease-associated phenotypes in multiple human cell types ► Minimal TALEN off-target effects, but significant clone-to-clone sequence variation Introduction: The study of human disease has been facilitated by the ability to identify the gene mutations responsible; at the same time, it has been hampered by the lack of an inexhaustible supply of easily accessible tissues from patients bearing those mutations. Another limitation is that many gene mutations that would be informative for disease biology if they could be studied in isolated cells are incompatible with human life (i.e., embryonic lethal). Classical gene-targeting technology via homologous recombination has proven to be an invaluable tool of experimental biology through its use in mouse embryonic stem cells for generating germline knockout and knockin mice; however, its use in mammalian systems has been limited primarily to studies in mice. In many cases, mice do not faithfully phenocopy human physiology and disease, e.g., cholesterol metabolism, coronary artery disease, and human hepatitis C virus (HCV) infection. The emergence of genome editing with engineered nucleases, as well as human pluripotent stem cell (hPSC) technology and differentiation protocols to obtain a variety of cell and tissue types in vitro, now make it possible to rapidly interrogate the effects of genetic modification in otherwise isogenic human model systems. Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that, due to their modular domain structure, have proven more straightforward to design and construct for performing genome editing than other types of nucleases (Bogdanove and Voytas, 2011). TALENs are typically designed as a pair that binds to genomic sequences flanking a target site and generates a double-strand break, which is repaired by the cell using either homology-directed repair (HDR) or the error-prone process of nonhomologous end-joining (NHEJ) (Christian et al., 2010; Li et al., 2011; Miller et al., 2011; Hockemeyer et al., 2011). NHEJ can be exploited to introduce small insertions or deletions (indels) resulting in frameshift mutations that effectively knock out a protein-coding gene. An exogenously introduced double-stranded DNA or single-stranded DNA oligonucleotide (ssODN) can serve as a repair template for HDR to incorporate an alteration into the genome (Soldner et al., 2011). In principle, TALEN pairs can be generated de novo with standard molecular biology techniques in a matter of days (Cermak et al., 2011; Sanjana et al., 2012). To demonstrate the utility, efficiency, and rapidity of TALEN technology in generating human cellular models with which to derive new biological insights, we created mutations in 15 genes and performed detailed phenotypic analysis of four genes for which novel roles in disease biology have emerged in recent years—APOB, SORT1, AKT2, and PLIN1. Results: Modular Assembly and Use of TALENs for Efficient and Rapid Genome Editing The DNA-binding domain of a TALEN comprises an array of 33- to 35-amino-acid monomers that are “coded” to recognize and bind specific DNA base pairs (bp) in a 1:1 fashion (Moscou and Bogdanove, 2009; Boch et al., 2009). We built upon previously described modular Golden Gate methodologies to allow assembly of multiple DNA fragments in an ordered fashion (Li et al., 2011; Cermak et al., 2011), such that a single ligation of preassembled tetramers and trimers generates TALENs that recognize any 15 bp recognition site in the genome (Figure 1; Figure S1 available online). This assembly method requires only 1–2 days for completion and is not prone to errors that complicate methods that rely on PCR amplification of monomers. Furthermore, we have developed a set of optimized vectors and methods for the delivery of TALENs into mammalian cells and, in particular, hPSCs. In brief, we transfect or electroporate TALEN pairs into cells and then subject them to fluorescence-activated cell sorting (FACS) 48 hr posttransfection based on fluorescent-marker expression. We replate the sorted cells at low density and allow them to recover and grow for 1 week, resulting in the formation of distinct single colonies. Colonies are expanded, genomic DNA purified, and mutations analyzed by PCR, agarose-gel screening, and Sanger sequencing (Figure 1 and Figure S1). The entire process from start to finish can be completed in less than one month., National Institutes of Health (U.S.) (R01-DK097768)

Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are new classes of genome-editing tools that target desired genomic sites in mammalian cells (Miller et al., 2011; Hockemeyer et al., 2011; Cong et al., 2013; Mali et al., 2013; Jinek et al., 2013). TALENs bind as a pair around a genomic site, in which a double-strand break (DSB) is introduced by a dimer of FokI nuclease domains. Recently published type II CRISPR/Cas systems use Cas9 nuclease that is targeted to a genomic site by complexing with a synthetic guide RNA that hybridizes a 20-nucleotide DNA sequence (“protospacer”) beginning with G and immediately preceding an NGG motif recognized by Cas9—constituting a G(N)19NGG target DNA sequence—resulting in a DSB three nucleotides upstream of the NGG motif (Jinek et al., 2012). However it is generated, the DSB instigates either non-homologous end-joining (NHEJ), which is error-prone and conducive to frameshift mutations (indels) that knock out gene alleles, or homology-directed repair (HDR), which can be exploited with the use of an exogenously introduced double-strand or single-strand DNA repair template to knock in or correct a mutation in the genome. We recently reported the use of a TALEN genome-editing system to rapidly and efficiently generate mutant alleles of 15 different genes in human pluripotent stem cells (hPSCs) as a means of performing rigorous disease modeling (Ding et al., 2013); the proportions of clones bearing at least one mutant alelle ranged from 2%–34%. Although one example of the use of CRISPRs in hPSCs has been reported (Mali et al., 2013), the efficiency of allele targeting was only 2%–4% (albeit in unsorted cells, in contrast to our system; see below). We sought to compare the relative efficacies of CRISPRs and TALENs targeting the same genomic sites in the same hPSC lines with the use of the same delivery platform as we described previously (Ding et al., 2013). In the TALEN genome-editing system, we used the CAG promoter to cotranslate (via a viral 2A peptide) each TALEN with green fluorescent protein (GFP) or red fluorescent protein (RFP). For CRISPRs, we subcloned a human codon-optimized Cas9 gene with a C-terminal nuclear localization signal (Mali et al., 2013) into the same CAG expression plasmid with GFP, and we separately expressed the guide RNA (gRNA) from a plasmid with the human U6 polymerase III promoter (Mali et al., 2013). The 20-nucleotide protospacer sequence for each gRNA was introduced using polymerase chain reaction (PCR)-based methods. Whether using TALENs or CRISPRs, equal amounts of the two plasmids were co-electroporated into hPSCs—either 25 μg of each plasmid, or 15 μg of each plasmid along with 30 μg of a DNA repair template if attempting knock-in—followed by fluorescence-activated cell sorting (FACS) after 24–48 hours, clonal expansion of single cells, and screening for mutations at the genomic target site via PCR. We designed gRNAs matching G(N)19NGG sequences in seven loci in six genes—AKT2, CELSR2, CIITA, GLUT4, LINC00116, and SORT1—that we had previously successfully targeted with TALENs (Ding et al., 2013) and one locus, in LDLR, that we had not. We found that in our system CRISPRs consistently and substantially outperformed TALENs across loci and hPSC lines (see Table S1). The TALENs yielded clones with at least one mutant allele at efficiencies of 0%–34%, but matched CRISPRs yielded mutant clones at efficiencies of 51%–79% (Table S1). Just as with TALENs, CRISPRs produced a variety of indels of sizes ranging from one nucleotide to several dozen nucleotides in size, centered on the predicted cleavage sites, suggesting that NHEJ mutagenesis occurs in the same way regardless of whether CRISPRs or TALENs are used. We also found that CRISPRs readily generated homozygous mutant clones (7%–25% of all clones; Table S1) as discerned by sequencing. We also attempted to knock in E17K mutations into AKT2 using a 67-nucleotide single-stranded DNA oligonucleotide as previously described (Ding et al., 2013). Although the predicted CRISPR cleavage site lay 11 and 13 nucleotides from the point mutations, respectively, the CRISPR yielded knock-in clones at a rate of 11%, whereas TALENs yielded only 1.6% (Table S1). We speculate that the superior performance of CRISPRs in our system is due to the Cas9 protein being more highly expressed and better tolerated than TALENs in hPSCs, as we routinely observed earlier (