Project description:Human iPSCs and NSCs were engineered by AAVS1 and/or C13 safe-harbor TALENs which mediated targeted integration of various reporter genes at single or dual safe-harbor loci. Multiple clones of targeted human iPSCs were used to compare with parental untargeted NCRM5 iPSCs. Polyclonal targeted human NSCs were used to compare with their parental untargeted NCRM1NSCs or H9NSCs. Total RNA obtained from targeted human iPSCs or NSCs compared to untargeted control iPSCs or NSCs.
Project description:Human iPSCs and NSCs were engineered by AAVS1 and/or C13 safe-harbor TALENs which mediated targeted integration of various reporter genes at single or dual safe-harbor loci. Multiple clones of targeted human iPSCs were used to compare with parental untargeted NCRM5 iPSCs. Polyclonal targeted human NSCs were used to compare with their parental untargeted NCRM1NSCs or H9NSCs.
Project description:X-CGD (X-linked chronic granulomatous disease) patient derived CD34+ cells were reprogrammed into two X-CGD iPSC clones (7 and 25) and subsequently corrected using ZFN (clone 7) and TALEN (clone 25) by integrating a therapeutic gp91phox expression cassette into the AAVS1 "safe harbor" locus Three condition experiment: before and after reprogramming and before and after genome editing.
Project description:X-CGD (X-linked chronic granulomatous disease) patient derived CD34+ cells were reprogrammed into two X-CGD iPSC clones (7 and 25) and subsequently corrected using ZFN (clone 7) and TALEN (clone 25) by integrating a therapeutic gp91phox expression cassette into the AAVS1 "safe harbor" locus
Project description:we identified gene-expression changes in induced pluripotent stem cells (iPSCs) and iPSC-derived neural stem cells (NSCs) from a patients with GLD (K-iPSCs/NSCs) and normal control (AF-iPSCs/NSCs), in order to investigate the potential mechanism underlying GLD pathogenesis. We identified 194 (K-iPSCs vs. AF-iPSCs) and 702 (K-NSCs vs. AF-NSCs) significantly dysregulated mRNAs when comparing the indicated groups. We also identified dozens of Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway terms that were enriched for the differentially expressed genes.
Project description:The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypic Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated for the first time the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
Project description:Bulk RNA-sequencing experiments were performed to analyze the transcriptomic effects of such integrations into two newly established genomic safe harbor sites. Jurkat and HEK293T cells were edited to integrate CMV-mRuby expressing cassette into Rogi2 genomic safe harbor site using Cas9 RNP
Project description:Fanconi Anemia (FA) is a recessive disorder associated with genomic instability We generated iPSC from FA patient fibroblasts and further corrected the mutated FANCA gene with a homologous recombination-based approach. The NSCs were differentiated from control-iPSCs, FA-iPSCs, and corrected FA-NSCs, and their gene expressions were determined by microarray analysis.
