2Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090 Novosibirsk, Russia
3Meshalkin National Medical Research Centre, Ministry of Healthcare of Russian Federation, 630090 Novosibirsk, Russia
4Novosibirsk State University, 630090 Novosibirsk, Russia
*To whom correspondence should be addressed.
Received April 30, 2019; Revised June 3, 2019; Accepted June 3, 2019
The CRISPR/Cas technology has a great potential in the treatment of many hereditary diseases. One of the prospective models for the CRISPR/Cas-mediated therapy is spinal muscular atrophy (SMA), a disease caused by deletion of the SMN1 gene that encodes the SMN protein required for the survival of motor neurons, SMA patients' genomes contain either single or several copies of SMN2 gene, which is a paralog of SMN1. Exon 7 of SMN2has the single-nucleotide substitution c.840C>T leading to the defective splicing and decrease in the amounts of the full-length SMN. The objective of this study was to create and test gene-editing systems for correction of the single-nucleotide substitution c.840C>T in exon 7 of theSMN2gene in fibroblasts, induced pluripotent stem cells, and motor neuron progenitors derived from a SMA patient. For this purpose, we used plasmid vectors expressing CRISPR/Cas9 and CRISPR/Cpf1, plasmid donor, and 90-nt single-stranded oligonucleotide templates that were delivered to the target cells by electroporation. Although sgRNA_T2 and sgRNA_T3 guiding RNAs were more efficient than sgRNA_T1 in fibroblasts (p < 0.05), no significant differences in the editing efficiency of sgRNA_T1, sgRNA_T2, and sgRNA_T3 was observed in patient-specific induced pluripotent stem cells and motor neuron progenitors. The highest editing efficiency in induced pluripotent stem cells and motor neuron progenitors was demonstrated by the sgRNA_T1 and single-stranded oligonucleotide donors.
KEY WORDS: spinal muscular atrophy, induced pluripotent stem cells, motor neuron progenitors, gene editing