Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

Renata M Martin; Kazuya Ikeda; M Kyle Cromer; Nobuko Uchida; Toshinobu Nishimura; Rosa Romano; Andrew J Tong; Viktor T Lemgart; Joab Camarena; Mara Pavel-Dinu; Camille Sindhu; Volker Wiebking; Sriram Vaidyanathan; Daniel P Dever; Rasmus O Bak; Anders Laustsen; Benjamin J Lesch; Martin R Jakobsen; Vittorio Sebastiano; Hiromitsu Nakauchi; Matthew H Porteus

doi:10.1016/j.stem.2019.04.001

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

Cell Stem Cell. 2019 May 2;24(5):821-828.e5. doi: 10.1016/j.stem.2019.04.001.

Authors

Renata M Martin¹, Kazuya Ikeda¹, M Kyle Cromer¹, Nobuko Uchida², Toshinobu Nishimura³, Rosa Romano¹, Andrew J Tong¹, Viktor T Lemgart¹, Joab Camarena¹, Mara Pavel-Dinu¹, Camille Sindhu¹, Volker Wiebking¹, Sriram Vaidyanathan¹, Daniel P Dever¹, Rasmus O Bak¹, Anders Laustsen⁴, Benjamin J Lesch¹, Martin R Jakobsen⁴, Vittorio Sebastiano⁵, Hiromitsu Nakauchi³, Matthew H Porteus⁶

Affiliations

¹ Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
² ReGen Med Division, BOCO Silicon Valley, Palo Alto, CA 94303, USA.
³ Department of Genetics, Stanford University, Stanford, CA 94305, USA.
⁴ Department of Biomedicine, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark; Aarhus Research Centre of Innate Immunology, Aarhus University, Wilhelm Meyers Alle 4, 8000 Aarhus C, Denmark.
⁵ Department of Obstetrics & Gynecology, Stanford University, Stanford, CA 94305, USA.
⁶ Department of Pediatrics, Stanford University, Stanford, CA 94305, USA. Electronic address: mporteus@stanford.edu.

PMID: 31051134
DOI: 10.1016/j.stem.2019.04.001

Abstract

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for in vitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

Keywords: AAV6; CRISPR/Cas9; ESC; RNP; electroporation; gene targeting; genome editing; homology-directed repair; iPSC; sgRNA.

Publication types

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

MeSH terms

CRISPR-Associated Protein 9 / genetics
CRISPR-Associated Protein 9 / metabolism*
CRISPR-Cas Systems / genetics*
Clustered Regularly Interspaced Short Palindromic Repeats
DNA Repair
Dependovirus / genetics*
Gene Editing / methods
Gene Frequency
Genetic Engineering
Genetic Vectors / genetics
Genotype*
Homologous Recombination
Humans
Pathology, Molecular
Pluripotent Stem Cells / physiology*
Tissue Donors

Substances

CRISPR-Associated Protein 9

Grants and funding

T32 GM007223/GM/NIGMS NIH HHS/United States