Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells.

Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.

Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.

Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.