Induced pluripotent stem cells (iPS cells) are immature cells generated from adult (mature) cells and have restored their ability to differentiate into any type of cell in the body. Induced pluripotent stem cells (iPS cells) are different from embryonic stem cells (ES cells), which form the internal cell mass of the embryo, but are also pluripotent and similar to primarycell, ultimately resulting in all cell types that make up the human body. Induced pluripotent cells were first described in 2006 by Japanese physician and researcher Shinya Yamanaka and colleagues. The initial experiment was performed using mouse cells. However, Yamanaka succeeded in obtaining iPS cells from human adult fibroblasts the following year. Before that, human stem cells could only be isolated from early human embryos. Therefore, an important feature of iPS cells is that their production does not require embryos, and the use of embryos is full of ethical issues.
Functional correction of chromosome inversion of large factor VIII gene in spontaneous iPSC of hemophilia patients using CRISPR-Cas9
Hemophilia A is an X-linked genetic disease caused by mutations in the F8 gene, which encodes coagulation factor VIII. Of all severe hemophilia A cases, almost half of the cases are caused by two total (140 kbp or 600 kbp) chromosome inversions, involving introns 1 and 22 of the F8 gene, respectively. Researchers derived induced pluripotent stem cells (iPSC) from patients with these reverse genotypes and used CRISPR-Cas9 nuclease to restore these chromosomal fragments to WT status. Based on whole-genome sequencing or targeted deep sequencing, the researchers isolated reverse-corrected iPSCs with a frequency of up to 6.7%, without detectable off-target mutations. In other deadly mouse models of hemophilia, endothelial cells differentiated from corrected iPSCs express the F8 gene and functionally rescue factor VIII deficiency. Therefore, the results provide a proof of principle for the functional correction of large chromosome rearrangements in patient-derived iPSCs, and propose potential therapeutic applications.
Using CRISPR-Cas9 to generate genetically corrected autologous iPSCs to treat hereditary retinal degeneration
Patient-derived induced pluripotent stem cells (iPSC) have broad prospects for autologous cell replacement. However, for many genetic diseases, treatment may require gene repair before transplantation. knockout cell line is very useful for this application. The purpose of this research is to develop a CRISPR-Cas9-mediated genome editing strategy to target and correct the three most common pathogenic types in patient-derived iPSCs: (1) exons, (2) deep inside Intron (3) dominant function. Researchers developed a homology-directed repair strategy for homozygous Alu insertion of male germ cell-associated kinase (MAK) exon 9, and demonstrated the reduction of retinal transcripts and proteins in patient cells. The researchers produced a CRISPR-Cas9-mediated non-homologous end joining (NHEJ) method to remove the main factor that causes Leber's congenital black disease, the IVS26 cryptographic mutation in CEP290, and proved the transcript in patient iPSC And protein correction. Finally, the researchers designed an allele-specific CRISPR guide that can selectively target Pro23His rhodopsin (RHO) mutant alleles. After delivering them to the patient's iPSC and the retina in vivo, a frameshift was generated. And premature termination can prevent the transcription of the disease-leading to variants. The strategies developed in this study will prove useful for correcting multiple genetic variations in genes that cause hereditary retinal degeneration.
Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility
Induced pluripotent stem cells (iPSCs) have strong potential in regenerative medicine applications; however, immune rejection caused by HLA mismatching is a concern. B2M gene knockout and HLA-homozygous iPSC stocks can address this issue, but the former approach may induce NK cell activity and fail to present antigens, and it is challenging to recruit rare donors for the latter method. Researcher show two genome-editing strategies for making immunocompatible donor iPSCs. First, researcher generated HLA pseudo-homozygous iPSCs with allele-specific editing of HLA heterozygous iPSCs. Second, researcher generated HLA-C-retained iPSCs by disrupting both HLA-A and -B alleles to suppress the NK cell response while maintaining antigen presentation. HLA-C-retained iPSCs could evade T cells and NK cells in vitro and in vivo. Researcher estimated that 12 lines of HLA-C-retained iPSCs combined with HLA-class II knockout are immunologically compatible with >90% of the world’s population, greatly facilitating iPSC-based regenerative medicine applications.
Make genome editing easier is the goal of Ubigene. We developed CRISPR-U™ (based on CRISPR/Cas9 technology) which is more efficient than general CRISPR/Cas9 in double-strand breaking, and CRISPR-U™ can greatly improve the efficiency of homologous recombination, easily achieve knockout (KO), point mutation (PM) and knockin (KI) in vitro and in vivo. With CRISPR-U™, Ubigene has successfully edit genes on more than 100 cell lines.
Park C Y, Kim D H, Son J S, et al. Functional Correction of Large Factor VIII Gene Chromosomal Inversions in Hemophilia A Patient-Derived iPSCs Using CRISPR-Cas9[J]. Cell Stem Cell, 2015, 17(2): 213-220.
Burnight E R, Gupta M, Wiley L A, et al. Using CRISPR-Cas9 to Generate Gene-Corrected Autologous iPSCs for the Treatment of Inherited Retinal Degeneration[J]. Molecular Therapy, 2017, 25(9): 1999-2013.
Xu H, Wang B, Ono M, et al. Targeted Disruption of HLA Genes via CRISPR-Cas9 Generates iPSCs with Enhanced Immune Compatibility[J]. Cell Stem Cell, 2019, 24(4).
The efficiency of gene knock-out and cleavage can not only give people the ability to generate protein radical profiles and establish regulatory records, but also has many advantages, making it a particularly attractive recombinant protein expression system. First, it is carboxylated on glutamic acid and sulfated on tyrosine. Second, the operation is simple, and the recombinant protein can be quickly produced through transient gene expression. Third, it can be used for stable recombinant protein production. Some researchers used gene cell knockout and cutting efficiency systems to generate gene-edited cell lines, targeted sequencing of GLUL genomic loci, produced stable EPO cell lines, and discovered the mechanism of stable expression of recombinant erythropoietin in humans .
According to customer needs, Yuanjing Biotechnology designs a stable gene transfer knockout program based on the target gene
Scheme 1: Small-segment gene knockout program, gRNA is set in the introns at both ends of exon 2, and the number of bases encoded by the knockout exon is not 3 times, and the knockout can cause frameshift.
Scheme 2: Frameshift gene knockout scheme, gRNA is set on the exon, the number of missing bases is not 3 times, and frameshift mutation can occur after knockout.
Scheme 3: Large-segment gene knockout scheme, knock out the coding sequence of the entire gene to achieve the effect of large-segment knockout.