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

TY - JOUR

T1 - Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination

AU - Martin, Renata M

AU - Ikeda, Kazuya

AU - Cromer, M Kyle

AU - Uchida, Nobuko

AU - Nishimura, Toshinobu

AU - Romano, Rosa

AU - Tong, Andrew J

AU - Lemgart, Viktor T

AU - Camarena, Joab

AU - Pavel-Dinu, Mara

AU - Sindhu, Camille

AU - Wiebking, Volker

AU - Vaidyanathan, Sriram

AU - Dever, Daniel P

AU - Bak, Rasmus O

AU - Laustsen, Anders

AU - Lesch, Benjamin J

AU - Jakobsen, Martin R

AU - Sebastiano, Vittorio

AU - Nakauchi, Hiromitsu

AU - Porteus, Matthew H

N1 - Copyright © 2019. Published by Elsevier Inc.

PY - 2019/5/2

Y1 - 2019/5/2

N2 - 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.

AB - 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.

KW - AAV6

KW - CRISPR/Cas9

KW - ESC

KW - RNP

KW - electroporation

KW - gene targeting

KW - genome editing

KW - homology-directed repair

KW - iPSC

KW - sgRNA

U2 - 10.1016/j.stem.2019.04.001

DO - 10.1016/j.stem.2019.04.001

M3 - Journal article

C2 - 31051134

SN - 1934-5909

VL - 24

SP - 821-828.e5

JO - Cell Stem Cell

JF - Cell Stem Cell

IS - 5

ER -