Synergistic gene editing in human iPS cells via cell cycle and DNA repair modulation.

Nature Communications
Thomas L Maurissen, Knut Woltjen

Abstract

Precise gene editing aims at generating single-nucleotide modifications to correct or model human disease. However, precision editing with nucleases such as CRIPSR-Cas9 has seen limited success due to poor efficiency and limited practicality. Here, we establish a fluorescent DNA repair assay in human induced pluripotent stem (iPS) cells to visualize and quantify the frequency of DNA repair outcomes during monoallelic and biallelic targeting. We found that modulating both DNA repair and cell cycle phase via defined culture conditions and small molecules synergistically enhanced the frequency of homology-directed repair (HDR). Notably, targeting in homozygous reporter cells results in high levels of editing with a vast majority of biallelic HDR outcomes. We then leverage efficient biallelic HDR with mixed ssODN repair templates to generate heterozygous mutations. Synergistic gene editing represents an effective strategy to generate precise genetic modifications in human iPS cells.

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Citations

Feb 18, 2021·ACS Synthetic Biology·Meng ZhangHuimin Zhao
Apr 8, 2021·Bioconjugate Chemistry·Daisuke Matsumoto, Wataru Nomura

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Methods Mentioned

BETA
fluorescence-activated cell sorting
genotyping
FACS
transfection
transgenic
Assay
PCR

Software Mentioned

LSM ZEN
FlowJo
ssODN
BD
MaxCyte
FACSDiva
ImageJ

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