Comparison of Zinc Finger Nucleases Versus CRISPR-Specific Nucleases for Genome Editing of the Wiskott-Aldrich Syndrome Locus

Abstract

Primary immunodeficiencies, including Wiskott-Aldrich syndrome (WAS), are a main target for genome-editing strategies using specific nucleases (SNs) because a small number of corrected hematopoietic stem cells could cure patients. In this work, we have designed various WAS gene-specific CRISPR/Cas9 systems and compared their efficiency and specificity with homodimeric and heterodimeric WAS-specific zinc finger nucleases (ZFNs), using K-562 cells as a cellular model and plasmid nucleofection or integration-deficient lentiviral vectors (IDLVs) for delivery. The various CRISPR/Cas9 and ZFN SNs showed similar efficiency when using plasmid nucleofection for delivery. However, dual IDLVs expressing ZFNs were more efficient than dual IDLVs expressing Cas9 and guide RNA or all-in-one IDLVs, expressing Cas9 and guide RNA in the same vector. The specificity of heterodimeric ZFNs and CRISPR/Cas9, measured by increments in γ-H2AX focus formation in WAS-edited cells, was similar for both, and both outperformed homodimeric ZFNs independently of the delivery system used. Interestingly, we show that delivery of SNs, using IDLVs, is more efficient and less genotoxic than plasmid nucleofection. We also show the similar behavior of heterodimeric ZFNs and CRISPR/Cas9 for homology-directed gene knock-in strategies, with 88 and 83% of the donors inserted in the WAS locus, respectively, whereas when using homodimeric ZFNs only 45% of the insertions were on target. In summary, our data indicate that CRISPR/Cas9 and heterodimeric ZFNs are both good alternatives to further develop SN-based gene therapy strategies for WAS. However, IDLV delivery of WAS-specific heterodimeric ZFNs was the best option of all systems compared in this study.

CRISPR-Cas, a robust gene-editing technology in the era of modern cancer immunotherapy

“…Unlike ZFN and TALEN that their target recognition is in demand of laborious protein design and synthesis for each target, CRISPR machinery is activated by guidance of a single RNA, sgRNA. Therefore, along with noticeable reduction in the time of design and construction, RNA leadership results in feasibility of CRISPR for multi-targeting, widespread genome manipulation or screening, and adoption in different biological contexts [ 49 , 50 ].…”

Nickel(ii)-promoted specific hydrolysis of zinc finger proteins

“…This is reflected in a number of recent publications. 65,[76][77][78] In addition the method described above can be generally applied for any engineered protein, and by optimizing the Ni(II)-induced protein hydrolysis to be significantly accelerated it may significantly contribute to this field. Most commonly occurring Zn(II) binding site donor set in ZF units.…”

Experimental DNA- or RNA-Directed Therapies for Trinucleotide Repeat Disease

“…A recent study showed similar behavior of heterodimeric zinc finger proteins and CRISPR/Cas9 for homology-directed gene knock-in strategies (88% and 83%, respectively, of the donors inserted in the target locus), whereas homodimeric zinc finger proteins showed only 45% on-target insertions [45].…”