Restoration of Dystrophin Expression in Mdx-Derived Muscle Progenitor Cells Using CRISPR/Cas9 System and Homology-Directed Repair Technology.

Duchenne muscular dystrophy (DMD) is a progressive myopathy caused by mutations in genes encoding dystrophin proteins that ultimately lead to depletion of myogenic progenitor cells (MPCs). Several approaches have been used to correctly express the dystrophin gene in induced pluripotent stem cells (iPSCs), including deletion of mutated exon 23 (ΔEx23) by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated gene 9 (Cas9)-mediated gene editing technology. However, this approach is labor-intensive due to individual colony picking and genotyping to verify allelic modification. Here, we present a protocol to restore the function of the dystrophin gene by using homology-directed repair (HDR)-based CRISPR/Cas9 and inducing myogenic program of reprogrammed iPSCs from Mdx mice by inducible muscle-specific transcription factor MyoD.

Krüppel-Like Factor 5 Regulates CFTR Expression Through Repression by Maintaining Chromatin Architecture Coupled with Direct Enhancer Activation.

Single cell RNA-sequencing has accurately identified cell types within the human airway that express the Cystic Fibrosis Transmembrane Conductance regulator (CFTR) gene. Low abundance CFTR transcripts are seen in many secretory cells, while high levels are restricted to rare pulmonary ionocytes. Here we focus on the mechanisms coordinating basal CFTR expression in the secretory compartment. Cell-selective regulation of CFTR is achieved within its invariant topologically associating domain by the recruitment of cis-regulatory elements (CREs). CRE activity is coordinated by cell-type-selective transcription factors. One such factor, Krüppel-Like Factor 5 (KLF5), profoundly represses CFTR transcript and protein in primary human airway epithelial cells and airway cell lines. Here we reveal the mechanism of action of KLF5 upon the CFTR gene. We find that depletion or ablation of KLF5 from airway epithelial cells changes higher order chromatin structure at the CFTR locus. Critical looping interactions that are required for normal gene expression are altered, the H3K27ac active chromatin mark is redistributed, and CTCF occupancy is modified. However, mutation of a single KLF5 binding site within a pivotal airway cell CRE abolishes CFTR expression. Hence, KLF5 has both direct activating and indirect repressive effects, which together coordinate CFTR expression in the airway.

Advances in Gene Therapy Techniques to Treat LRRK2 Gene Mutation.

Leucine-rich repeat kinase 2 (LRRK2) gene mutation is an autosomal dominant mutation associated with Parkinson's disease (PD). Among LRRK2 gene mutations, the LRRK2 G2019S mutation is frequently involved in PD onset. Currently, diverse gene correction tools such as zinc finger nucleases (ZFNs), helper-dependent adenoviral vector (HDAdV), the bacterial artificial chromosome-based homologous recombination (BAC-based HR) system, and CRISPR/Cas9-homology-directed repair (HDR) or adenine base editor (ABE) are used in genome editing. Gene correction of the LRRK2 G2019S mutation has been applied whenever new gene therapy tools emerge, being mainly applied to induced pluripotent stem cells (LRRK2 G2019S-mutant iPSCs). Here, we comprehensively introduce the principles and methods of each programmable nuclease such as ZFN, CRISPR/Cas9-HDR or ABE applied to LRRK2 G2019S, as well as those of HDAdV or BAC-based HR systems used as nonprogrammable nuclease systems.