Abstract
Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template.
Highlights
Homologous recombination in hESCs mediated gene targeting with wild type Cas9 that enables homozygous targeting of both alleles, no homozygous knock-in clones were identified in this study
To achieve homozygous knock-in clones with long DNA templates, a “double nicking” strategy might be employed with paired offset guide RNAs to significantly increase the homozygous targeting efficiency (Ran et al, 2013)
We found three singlecrossover clones with homologous recombination occurred only at the 5′ homologous arm
Summary
Homologous recombination in hESCs mediated gene targeting with wild type Cas9 that enables homozygous targeting of both alleles, no homozygous knock-in clones were identified in this study. To achieve homozygous knock-in clones with long DNA templates, a “double nicking” strategy might be employed with paired offset guide RNAs to significantly increase the homozygous targeting efficiency (Ran et al, 2013).
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