技术资料

CRISPR/Cas9 has demonstrated a high-efficiency in site-specific gene targeting. However,potential off-target effects of the Cas9 nuclease represent a major safety concern for any therapeutic application. Here,we knock out the Tafazzin gene by CRISPR/Cas9 in human-induced pluripotent stem cells with 54% efficiency. We combine whole-genome sequencing and deep-targeted sequencing to characterise the off-target effects of Cas9 editing. Whole-genome sequencing of Cas9-modified hiPSC clones detects neither gross genomic alterations nor elevated mutation rates. Deep sequencing of in silico predicted off-target sites in a population of Cas9-treated cells further confirms high specificity of Cas9. However,we identify a single high-efficiency off-target site that is generated by a common germline single-nucleotide variant (SNV) in our experiment. Based on in silico analysis,we estimate a likelihood of SNVs creating off-target sites in a human genome to be ˜1.5-8.5%,depending on the genome and site-selection method,but also note that mutations might be generated at these sites only at low rates and may not have functional consequences. Our study demonstrates the feasibility of highly specific clonal ex vivo gene editing using CRISPR/Cas9 and highlights the value of whole-genome sequencing before personalised CRISPR design. View Publication

Nature Genetics 47,469 (2015). doi:10.1038/ng.3258 View Publication

BACKGROUND: Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been successfully used to knock out endogenous genes in stem cell research. However,the deficiencies of current gene-based delivery systems may hamper the clinical application of these nucleases. A new delivery method that can improve the utility of these nucleases is needed.backslashnbackslashnRESULTS: In this study,we utilized a cell-penetrating peptide-based system for ZFN and TALEN delivery. Functional TAT-ZFN and TAT-TALEN proteins were generated by fusing the cell-penetrating TAT peptide to ZFN and TALEN,respectively. However,TAT-ZFN was difficult to purify in quantities sufficient for analysis in cell culture. Purified TAT-TALEN was able to penetrate cells and disrupt the gene encoding endogenous human chemokine (C-C motif) receptor 5 (CCR5,a co-receptor for HIV-1 entry into cells). Hypothermic treatment greatly enhanced the TAT-TALEN-mediated gene disruption efficiency. A 5% modification rate was observed in human induced pluripotent stem cells (hiPSCs) treated with TAT-TALEN as measured by the Surveyor assay.backslashnbackslashnCONCLUSIONS: TAT-TALEN protein-mediated gene disruption was applicable in hiPSCs and represents a promising technique for gene knockout in stem cells. This new technique may advance the clinical application of TALEN technology. View Publication

The human induced pluripotent stem cell (hiPSC) provides a breakthrough approach that helps overcoming ethical and allergenic challenges posed in application of neural stem cells (NSCs) in targeted cancer gene therapy. However,the tumor-tropic capacity of hiPSC-derived NSCs (hiPS-NSCs) still has much room to improve. Here we attempted to promote the tumor tropism of hiPS-NSCs by manipulating the activity of endogenous miR-199a/214 cluster that is involved in regulation of hypoxia-stimulated cell migration. We first developed a baculovirus-delivered CRISPR interference (CRISPRi) system that sterically blocked the E-box element in the promoter of the miR-199a/214 cluster with an RNA-guided catalytically dead Cas9 (dCas9). We then applied this CRISPRi system to hiPS-NSCs and successfully suppressed the expression of miR-199a-5p,miR-199a-3p,and miR-214 in the microRNA gene cluster. Meanwhile,the expression levels of their targets related to regulation of hypoxia-stimulated cell migration,such as HIF1A,MET,and MAPK1,were upregulated. Further migration assays demonstrated that the targeted inhibition of the miR-199a/214 cluster significantly enhanced the tumor tropism of hiPS-NSCs both in vitro and in vivo. These findings suggest a novel application of CRISPRi in NSC-based tumor-targeted gene therapy. View Publication

Hemophilia A,one of the most common genetic bleeding disorders,is caused by various mutations in the blood coagulation factor VIII (F8) gene. Among the genotypes that result in hemophilia A,two different types of chromosomal inversions that involve a portion of the F8 gene are most frequent,accounting for almost half of all severe hemophilia A cases. In this study,we used a transcription activator-like effector nuclease (TALEN) pair to invert a 140-kbp chromosomal segment that spans the portion of the F8 gene in human induced pluripotent stem cells (iPSCs) to create a hemophilia A model cell line. In addition,we reverted the inverted segment back to its normal orientation in the hemophilia model iPSCs using the same TALEN pair. Importantly,we detected the F8 mRNA in cells derived from the reverted iPSCs lines,but not in those derived from the clones with the inverted segment. Thus,we showed that TALENs can be used both for creating disease models associated with chromosomal rearrangements in iPSCs and for correcting genetic defects caused by chromosomal inversions. This strategy provides an iPSC-based novel therapeutic option for the treatment of hemophilia A and other genetic diseases caused by chromosomal inversions. View Publication

The available techniques for directed gene manipulation in the mouse are unprecedented in any multicellular organism and make the mouse an invaluable tool for unraveling all aspects of mammalian biology. To realize fully the potential of these genetic tools requires that phenotypic analysis be efficient,rapid,and complete. Genetic chimeras and mosaics,in which mutant cells are mixed with wild-type cells,can be used to augment standard analysis of intact mutant animals and alleviate the time required and the expense involved in generating and maintaining multiple strains of mutant mice. View Publication

X-linked chronic granulomatous disease (X-CGD) is an immune deficiency resulting from defective production of microbicidal reactive oxygen species (ROS) by phagocytes. Causative mutations occur throughout the CYBB gene,resulting in absent or defective gp91(phox) protein expression. To correct CYBB exon 5 mutations while retaining normal gene regulation,we utilized TALEN or Cas9 for exon 5 replacement in induced pluripotent stem cells (iPSCs) from patients,which restored gp91(phox) expression and ROS production in iPSC-derived granulocytes. Alternate approaches for correcting the majority of X-CGD mutations were assessed,involving TALEN- or Cas9-mediated insertion of CYBB minigenes at exon 1 or 2 of the CYBB locus. Targeted insertion of an exon 1-13 minigene into CYBB exon 1 resulted in no detectable gp91(phox) expression or ROS activity in iPSC-derived granulocytes. In contrast,targeted insertion of an exon 2-13 minigene into exon 2 restored both gp91(phox) and ROS activity. This demonstrates the efficacy of two correction strategies: seamless repair of specific CYBB mutations by exon replacement or targeted insertion of an exon 2-13 minigene to CYBB exon 2 while retaining exon/intron 1. Furthermore,it highlights a key issue for targeted insertion strategies for expression from an endogenous promoter: retention of intronic elements can be necessary for expression. View Publication

Background The AAVS1 locus is viewed as a ‘safe harbor' for transgene insertion into human genome. In the present study,we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected,stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting,25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette,we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency,stability in expression of inserted transgene and flexibility in transgene exchange,the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research,drug development,regenerative medicine and gene therapy. Copyright textcopyright 2013 John Wiley & Sons,Ltd. View Publication

The in vitro differentiation of human induced pluripotent stem cells (hiPSC) to generate specific types of cells is inefficient,and the remaining undifferentiated cells may form teratomas. This raises safety concerns for clinical applications of hiPSC-derived cellular products. To improve the safety of hiPSC,we attempted to site-specifically insert a herpes simplex virus 1 thymidine kinase (HSV1-TK) suicide gene at the endogenous OCT4 (POU5F1) locus of hiPSC. Since the endogenous OCT4 promoter is active in undifferentiated cells only,we speculated that the HSV1-TK suicide gene will be transcribed in undifferentiated cells only and that the remaining undifferentiated cells can be depleted by treating them with the prodrug ganciclovir (GCV) prior to transplantation. To insert the HSV1-TK gene at the OCT4 locus,we cotransfected hiPSC with a pair of plasmids encoding an OCT4-specific zinc finger nuclease (ZFN) and a donor plasmid harboring a promoter-less transgene cassette consisting of HSV1-TK and puromycin resistance gene sequences,flanked by OCT4 gene sequences. Puromycin resistant clones were established and characterized regarding their sensitivity to GCV and the site of integration of the HSV1-TK/puromycin resistance gene cassette. Of the nine puromycin-resistant iPSC clones analyzed,three contained the HSV1-TK transgene at the OCT4 locus,but they were not sensitive to GCV. The other six clones were GCV-sensitive,but the TK gene was located at off-target sites. These TK-expressing hiPSC clones remained GCV sensitive for up to 90 days,indicating that TK transgene expression was stable. Possible reasons for our failed attempt to selectively target the OCT4 locus are discussed. View Publication

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS),as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear,with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed,and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased,leading to accumulation of GGGGCC repeat-containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-α,suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6,and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. View Publication