Stem cells are undifferentiated biological cells that can differentiate into specialized cells and can divide (through mitosis) to produce more stem cells. They exist in multicellular organisms. In mammals, there are two types of stem cells: embryonic stem cells isolated from the inner cell mass of blastocysts and primary cells found in various tissues. In adult organisms, stem cells and primarycells can be used as the body's repair system to supplement adult tissues. In the developing embryo, stem cells can differentiate into all specialized cells-ectoderm, endoderm and mesoderm-but can maintain the normal turnover of regenerated organs (such as blood, skin or intestinal tissue).
Precision medicine: genetic repair of retinitis pigmentosa in stem cells derived from patients
Induced pluripotent stem cells (iPSC) derived from patient fibroblasts may be used as a source of autologous cells for transplantation of retinal diseases. However, patient-derived iPSCs still carry pathogenic mutations. In order to generate healthy patient-derived cells, a new gene editing technology based on the bacterial system of short palindrome repeats (CRISPR)/Cas9 distributed in clusters can be used to repair mutations, thereby producing grafts that do not require patient immunosuppression. The researchers tested whether CRISPR/Cas9 can be used in patient-specific iPSCs to precisely repair the RPGR point mutation that causes X-linked retinitis pigmentosa (XLRP). Fibroblasts cultured from needle biopsies of XLRP patients are transduced to produce iPSCs with the patient's c.3070G>T mutation. Use CRISPR guide RNA, Cas9 endonuclease and donor homology template to transduce iPSC. Although the gene has repetitive sequences and GC-rich sequences, 13% of RPGR gene copies show mutation correction and conversion to wild-type alleles. This is the first report of using CRISPR to correct pathogenic mutations in iPSCs from patients with photoreceptor degeneration. This important proof-of-concept discovery supports the development of personalized iPSC-based transplant therapy for retinal diseases.
CRISPR/Cas9β-globin gene targets human hematopoietic stem cells
Beta-hemoglobinopathy, such as sickle cell disease and beta-thalassemia, is caused by mutations in the beta-globin (HBB) gene and affects millions of people worldwide. Researchers have proposed a CRISPR/Cas9 gene editing system, which combines Cas9 ribonucleoprotein and adeno-associated virus vector homologous donors to transplant human hematopoietic stem cells, which can carry out in vitro genes in patient-derived hematopoietic stem cells Correction and then autotransplant. Homologous recombination is achieved at the HBB gene of hematopoietic stem cells. It is worth noting that the researchers designed an enrichment model to purify hematopoietic stem cells and progenitor cells with more than 90% targeted integration. The researchers also showed that by using patient-derived stem cells and progenitor cells, they can differentiate into red blood cells and express adult β-globulin (HbA) messenger RNA, thereby effectively correcting the Glu6Val mutation that causes sickle cell disease. This confirms Complete transcriptional regulation of edited HBB alleles. In summary, these preclinical studies outline a CRISPR-based approach that targets hematopoietic stem cells through homologous recombination at the HBB site, thereby promoting the development of next-generation therapies for β-hemoglobinopathy.
Repair of CRISPR–Cas9-induced double-strand breaks leads to large deletions and complex rearrangements
CRISPR–Cas9 is expected to become the preferred gene editing tool in clinical settings. So far, the exploration of Cas9-induced genetic changes is limited to the proximal and long-distance off-target sequences of the target site, and it is concluded that CRISPR-Cas9 has reasonable specificity. Researchers reported major target mutagenesis, such as mouse embryonic stem cells, mouse hematopoietic progenitor cells, and human differentiated cell lines with large deletions of target sites and more complex genome rearrangements. Researchers used long-read sequencing and long-distance PCR genotyping methods and found that DNA breaks introduced by single guide RNA/Cas9 are often resolved as multiple base deletions. In addition, lesions and crossover events at the distal end of the incision site were identified. The observed genome damage in mitotically active cells caused by CRISPR–Cas9 editing may be pathogenic.
CRISPR-U™ (based on CRISPR/Cas9 technology) developed by Ubigene is more effective than ordinary CRISPR/Cas9 in double-strand breaks, and CRISPR-U™ can greatly improve the efficiency of homologous recombination and easily achieve knock-out (KO)) , Point mutation (PM) and knock-in (KI) in vitro and in vivo. With CRISPR-U, Ubigene has successfully edited genes on more than 100 cell lines.
Bassuk, A., Zheng, A., Li, Y. et al. Precision Medicine: Genetic Repair of Retinitis Pigmentosa in Patient-Derived Stem Cells. Sci Rep 6, 19969 (2016). https://doi.org/10.1038/srep19969
Dever, D., Bak, R., Reinisch, A. et al. CRISPR/Cas9 β-globin gene targeting in human haematopoietic stem cells. Nature 539, 384–389 (2016). https://doi.org/10.1038/nature20134
Kosicki, M., Tomberg, K. & Bradley, A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements. Nat Biotechnol 36, 765–771 (2018). https://doi.org/10.1038/nbt.4192
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.