We have generated an induced pluripotent stem cell (iPSC) line, IGIBi001-A from an Indian sickle cell patient with a homozygous HBB gene mutation using Sendai virus reprogramming system.
We took a combinatorial approach using single-stranded oligonucleotide and a CRISPR/Cas9 ribonucleoprotein to convert wild-type HBB into the sickle cell genotype by evaluating conditions for two common delivery strategies of gene editing tools into CD34+ cells.
Here, we demonstrate that Transcription Activator-Like Effector Nucleases (TALENs) and the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 nuclease system can target DNA sequences around the sickle-cell mutation in the β-globin gene for site-specific cleavage and facilitate precise correction when a homologous donor template is codelivered.
We explore RBC storage stability and post-transfusion recovery in murine models of allogeneic and xenogeneic transfusion using blood from humanized transgenic sickle cell hemizygous mice (Hba<sup>tm1Paz</sup>Hbb<sup>tm1Tow</sup>Tg(HBA-HBBs)41Paz/J) and human donors with a common genetic mutation sickle cell trait (HbAS).
Several hemoglobinopathies result from homozygosity or compound heterozygosity to beta-globin (HBB) gene mutations, such as that producing sickle cell hemoglobin (HbS), HbC, HbD and HbE.
Using a psoralen delivery system mediated by a DNA third strand that binds selectively to linear target duplexes immediately downstream from the Sickle Cellbeta-globin gene mutation and the comparable wild-type beta-globin gene sequence, the kinetics of formation and yield of psoralen monoadducts and crosslinks with pyrimidine residues at and near the mutant base pair site and its wild-type counterpart were determined.
Three identical deoxyoligonucleotide third strands with a 3'-terminal psoralen moiety attached by linkers that differ in length (N = 16, 6 and 4 atoms) and structure were examined for their ability to form triplex-directed psoralen photoproducts with both the mutant T residue of the Sickle Cellbeta-globin gene and the comparable wild-type sequence in linear duplex targets.
The potential and reliability of DNA analysis for the identification of human remains are demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia. beta-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved residual DNA.
To investigate the role of the alpha-globin gene microdeletion and beta-globin gene cluster haplotypes on the degree of glomerular involvement, 76 adult SSA patients (hemoglobin SS) were studied to determine the relationship between these genetic markers and the development of sickle cell glomerulopathy.
Efficient polymerase chain reaction (PCR) amplification of a 680 base pair sequence of the beta-globin gene spanning the site of the sickle cell mutation was obtained for 79 per cent of single heterozygous cells.
The targeting system studied was designed to correct a human sickle-cell beta-globin-encoding gene (HBBS) on human chromosome 11 by replacing the defective gene with incoming DNA carrying normal HBB sequences.
Structural analysis of the 5' flanking region of the beta-globin gene in African sickle cell anemia patients: further evidence for three origins of the sickle cell mutation in Africa.