Nuclease-Mediated Double-Strand Break (DSB) Enhancement of Small Fragment Homologous Recombination (SFHR) Gene Modification in Human-Induced Pluripotent Stem Cells (hiPSCs)

Abstract

Recent developments in methods to specifically modify genomic DNA using sequence-specific endonucleases and donor DNA have opened the door to a new therapeutic paradigm for cell and gene therapy of inherited diseases. Sequence-specific endonucleases, in particular transcription activator-like (TAL) effector nucleases (TALENs), have been coupled with polynucleotide small/short DNA fragments (SDFs) to correct the most common mutation in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, a 3-base-pair deletion at codon 508 (delF508), in induced pluripotent stem (iPS) cells. The studies presented here describe the generation of candidate TALENs and their co-transfection with wild-type (wt) CFTR-SDFs into CF-iPS cells homozygous for the delF508 mutation. Using an allele-specific PCR (AS-PCR)-based cyclic enrichment protocol, clonal populations of corrected CF-iPS cells were isolated and expanded.

Open-door versus French-door laminoplasty for the treatment of cervical multilevel compressive myelopathy

“…Since cervical laminoplasty was first introduced by Oyama and Moriwaki with the name of ''expansive Z-laminoplasty'' in 1972 [8], various types of cervical laminoplasty have been developed. At present, the surgical methods of cervical laminoplasty are classified into two types, namely open-door laminoplasty (ODL) and French-door laminoplasty (FDL) (Fig.…”

CRISPR/Cas9-Mediated Three Nucleotide Insertion Corrects a Deletion Mutation in MRP1/ABCC1 and Restores Its Proper Folding and Function

“…The correction of three nucleotide deletion in CFTR-ΔF508 has been achieved in either induced pluripotent stem cells42, 43, 44 or cystic fibrosis patient-derived organoids 38 . However, modification of the ends generated by DSB and the NHEJ-mediated mutation is still a big issue for genome editing 41 .…”

Fixing Stem Cells Via Genome Editing: Hope for Cystic Fibrosis?

“…The design and assessment of CFTR-specific nucleases also has been reported previously, including repair of the mutant CFTR gene [6][7][8][9][10]. There are now several classes of sequence-specific nucleases available (ZFNs, TALENs, CRISPR/Cas9, Meganucleases) for DNA sequence-specific gene editing.…”

“…The generation of iPSCs from CF patients has been reported previously [3][4][5][6], with subsequent differentiation into epithelial cells. The design and assessment of CFTR-specific nucleases also has been reported previously, including repair of the mutant CFTR gene [6][7][8][9][10].…”