Microhomology-assisted scarless genome editing in human iPSCs

Shin Il Kim, Tomoko Matsumoto, Harunobu Kagawa, Michiko Nakamura, Ryoko Hirohata, Ayano Ueno, Maki Ohishi, Tetsushi Sakuma, Tomoyoshi Soga, Takashi Yamamoto, Knut Woltjen

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

Gene-edited induced pluripotent stem cells (iPSCs) provide relevant isogenic human disease models in patient-specific or healthy genetic backgrounds. Towards this end, gene targeting using antibiotic selection along with engineered point mutations remains a reliable method to enrich edited cells. Nevertheless, integrated selection markers obstruct scarless transgene-free gene editing. Here, we present a method for scarless selection marker excision using engineered microhomology-mediated end joining (MMEJ). By overlapping the homology arms of standard donor vectors, short tandem microhomologies are generated flanking the selection marker. Unique CRISPR-Cas9 protospacer sequences nested between the selection marker and engineered microhomologies are cleaved after gene targeting, engaging MMEJ and scarless excision. Moreover, when point mutations are positioned unilaterally within engineered microhomologies, both mutant and normal isogenic clones are derived simultaneously. The utility and fidelity of our method is demonstrated in human iPSCs by editing the X-linked HPRT1 locus and biallelic modification of the autosomal APRT locus, eliciting disease-relevant metabolic phenotypes.

Original languageEnglish
Article number939
JournalNature Communications
Volume9
Issue number1
DOIs
Publication statusPublished - 2018 Dec 1

Fingerprint

Induced Pluripotent Stem Cells
editing
stem cells
genome
Stem cells
Gene Targeting
Genes
genes
markers
Point Mutation
Clustered Regularly Interspaced Short Palindromic Repeats
Joining
loci
mutations
Metabolic Diseases
metabolic diseases
Transgenes
Clone Cells
phenotype
Tissue Donors

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physics and Astronomy(all)

Cite this

Kim, S. I., Matsumoto, T., Kagawa, H., Nakamura, M., Hirohata, R., Ueno, A., ... Woltjen, K. (2018). Microhomology-assisted scarless genome editing in human iPSCs. Nature Communications, 9(1), [939]. https://doi.org/10.1038/s41467-018-03044-y

Microhomology-assisted scarless genome editing in human iPSCs. / Kim, Shin Il; Matsumoto, Tomoko; Kagawa, Harunobu; Nakamura, Michiko; Hirohata, Ryoko; Ueno, Ayano; Ohishi, Maki; Sakuma, Tetsushi; Soga, Tomoyoshi; Yamamoto, Takashi; Woltjen, Knut.

In: Nature Communications, Vol. 9, No. 1, 939, 01.12.2018.

Research output: Contribution to journalArticle

Kim, SI, Matsumoto, T, Kagawa, H, Nakamura, M, Hirohata, R, Ueno, A, Ohishi, M, Sakuma, T, Soga, T, Yamamoto, T & Woltjen, K 2018, 'Microhomology-assisted scarless genome editing in human iPSCs', Nature Communications, vol. 9, no. 1, 939. https://doi.org/10.1038/s41467-018-03044-y
Kim SI, Matsumoto T, Kagawa H, Nakamura M, Hirohata R, Ueno A et al. Microhomology-assisted scarless genome editing in human iPSCs. Nature Communications. 2018 Dec 1;9(1). 939. https://doi.org/10.1038/s41467-018-03044-y
Kim, Shin Il ; Matsumoto, Tomoko ; Kagawa, Harunobu ; Nakamura, Michiko ; Hirohata, Ryoko ; Ueno, Ayano ; Ohishi, Maki ; Sakuma, Tetsushi ; Soga, Tomoyoshi ; Yamamoto, Takashi ; Woltjen, Knut. / Microhomology-assisted scarless genome editing in human iPSCs. In: Nature Communications. 2018 ; Vol. 9, No. 1.
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