Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells

Hum Gene Ther. 2018 Nov;29(11):1252-1263. doi: 10.1089/hum.2017.255. Epub 2018 May 9.

Abstract

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

Keywords: CRISPR/Cpf1; gene conversion; iPSCs; motor neurons; spinal muscular atrophy.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Differentiation
  • Clone Cells
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gene Conversion*
  • Genotype
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Male
  • Motor Neurons / metabolism
  • Muscular Atrophy, Spinal / genetics*
  • Oligodeoxyribonucleotides / metabolism*
  • Proto-Oncogene Proteins c-myc / metabolism
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Survival of Motor Neuron 1 Protein / genetics*
  • Survival of Motor Neuron 2 Protein / genetics*
  • Young Adult

Substances

  • Oligodeoxyribonucleotides
  • Proto-Oncogene Proteins c-myc
  • RNA, Guide, CRISPR-Cas Systems
  • Survival of Motor Neuron 1 Protein
  • Survival of Motor Neuron 2 Protein