CRISPR-MAD7 Genomic Safe Harbor Sites
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ABSTRACT:
PROVIDER: PRJEB67344 | ENA |
REPOSITORIES: ENA
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Project description:One common strategy in gene and cell therapies requires the integration and stable transcription of transgenes into the host cell genomes. Genomic safe harbors (GSHs) are regions of the genome that can maintain the expression of transgenes without disrupting the transcriptome of host cells. With recent advances in genome editing technology, GSHs play an increasingly important role in improving the efficiency and safety of genome engineering. However, limited candidate GSHs have been identified. In addition, many parameters currently used to identify GSHs are not knowledge based and none of the current available GSHs meet all the ideal safety requirement. In this study, we use the polymorphic mobile element insertions (pMEIs) that naturally occur among healthy human populations to facilitate GSHs searching. By integrating pMEIs with epigenomic signatures and 3D chromatin organization information, we developed a framework to map cell typespecific GSHs. We applied our framework to pMEIs identified in the 1000 Genomes project and GTEX projects and identified novel GSHs Candidates in blood and brain cells. We further validated that a transgene cassette integrated in one novel GSH can be stably transcribed without affecting normal proliferation and differentiation of host cells. We also developed a user-friendly program to search for GSHs in different population and cell types.
2021-09-13 | GSE183935 | GEO
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
2022-02-11 | E-MTAB-11289 | biostudies-arrayexpress
Project description:Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent, broadly applicable, while minimizing risks of off-target integration. In the liver, the Albumin locus is currently the only well characterized site for promoterless transgene insertion. Using an unbiased ChIP-seq approach, we here identify Apolipoprotein a1 (Apoa1) as one of the most highly expressed and accessible loci in mouse and human liver. We target the Apoa1 locus with Adeno-Associated Viral (AAV) delivery of CRISPR/Cas9, and achieve rates of 6 to 16% with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins such as factor IX (FIX) or apolipoprotein E (APOE). Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as novel safe harbor site for genome editing therapeutics.
2021-06-01 | GSE152993 | GEO
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.
2014-09-01 | E-GEOD-55975 | biostudies-arrayexpress
Project description:Identification of Genomic Safe Harbors in the Anhydrobiotic Cell Line, Pv11
| PRJNA787263 | ENA
Project description:Transcriptomes of novel human genomic safe harbor sites for gene and cell therapies
| PRJEB50576 | ENA
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.
2014-09-01 | GSE55975 | GEO
Project description:The advent of human induced pluripotent stem (iPS) cells enables for the first time the derivation of unlimited numbers of patient-specific stem cells and holds great promise for regenerative medicine. However, realizing the full potential of iPS cells requires robust, precise and safe strategies for their genetic modification. Safe human iPS cell engineering is especially needed for therapeutic applications, as stem cell-based therapies that rely on randomly integrated transgenes pose oncogenic risks. Here we describe a strategy to genetically modify iPS cells from patients with beta-thalassemia in a potentially clinically relevant manner. Our approach is based on the identification and selection of âsafe harborâ sites for transgene expression in the human genome. We show that thalassemia patient iPS cell clones harboring a transgene can be isolated and screened according to chromosomal position. We next demonstrate that iPS cell clones that meet our âsafe harborâ criteria resist silencing and allow for therapeutic levels of beta-globin expression upon erythroid differentiation without perturbation of neighboring gene expression. Combined bioinformatics and functional analyses thus provide a robust and dependable approach for achieving desirable levels of transgene expression from selected chromosomal loci. This approach may be broadly applicable to introducing therapeutic or suicide genes into patient specific iPS cells for use in cell therapy. iPS cell clones were derived from beta-thalassemia patients. A single copy of beta-globin transgene cis-linked to locus control region (LCR) elements and an excisable Neo-eGFP transcription unit were inserted into these cell clones. beta-globin expression was induced by erythroid differentiation.
2010-10-26 | E-GEOD-24901 | biostudies-arrayexpress
Project description:Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.
| S-EPMC4587366 | biostudies-literature
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.
2024-02-09 | GSE249731 | GEO