Efficient and allele-specific genome editing of disease loci in human iPSCs

Mol Ther. 2015 Mar;23(3):570-7. doi: 10.1038/mt.2014.226. Epub 2014 Nov 24.

Abstract

Efficient and precise genome editing is crucial for realizing the full research and therapeutic potential of human induced pluripotent stem cells (iPSCs). Engineered nucleases including CRISPR/Cas9 and transcription activator like effector nucleases (TALENs) provide powerful tools for enhancing gene-targeting efficiency. In this study, we investigated the relative efficiencies of CRISPR/Cas9 and TALENs in human iPSC lines for inducing both homologous donor-based precise genome editing and nonhomologous end joining (NHEJ)-mediated gene disruption. Significantly higher frequencies of NHEJ-mediated insertions/deletions were detected at several endogenous loci using CRISPR/Cas9 than using TALENs, especially at nonexpressed targets in iPSCs. In contrast, comparable efficiencies of inducing homologous donor-based genome editing were observed at disease-associated loci in iPSCs. In addition, we investigated the specificity of guide RNAs used in the CRISPR/Cas9 system in targeting disease-associated point mutations in patient-specific iPSCs. Using myeloproliferative neoplasm patient-derived iPSCs that carry an acquired JAK2-V617F point mutation and α1-antitrypsin (AAT) deficiency patient-derived iPSCs that carry an inherited Z-AAT point mutation, we demonstrate that Cas9 can specifically target either the mutant or the wild-type allele with little disruption at the other allele differing by a single nucleotide. Overall, our results demonstrate the advantages of the CRISPR/Cas9 system in allele-specific genome targeting and in NHEJ-mediated gene disruption.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Sequence
  • CRISPR-Associated Protein 9
  • Cell Line
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA End-Joining Repair
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Expression
  • Genome, Human*
  • Humans
  • Induced Pluripotent Stem Cells / metabolism*
  • Induced Pluripotent Stem Cells / pathology
  • Janus Kinase 2 / genetics
  • Janus Kinase 2 / metabolism
  • Molecular Sequence Data
  • Mutation
  • Myeloproliferative Disorders / genetics
  • Myeloproliferative Disorders / metabolism
  • Myeloproliferative Disorders / pathology
  • Quantitative Trait Loci*
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • RNA, Guide, CRISPR-Cas Systems / metabolism
  • Recombinational DNA Repair
  • alpha 1-Antitrypsin / genetics
  • alpha 1-Antitrypsin / metabolism

Substances

  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • alpha 1-Antitrypsin
  • Janus Kinase 2
  • CRISPR-Associated Protein 9
  • Cas9 protein, Francisella novicida
  • Endonucleases