Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template

Rong, Zhili; Zhu, Shengyun; Xu, Yang; Fu, Xuemei

doi:10.1007/s13238-014-0032-5

Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template

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Published: 14 March 2014

Volume 5, pages 258–260, (2014)
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Protein & Cell

Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template

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Zhili Rong²,
Shengyun Zhu¹,
Yang Xu² &
…
Xuemei Fu^1,3

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Dear Editor,

Genome editing of human embryonic stem cells (hESCs) is critical for basic biological research and regenerative medicine. However, until a few years ago, gene targeting technologies to disrupt, repair or overexpress genes in hESCs had been very inefficient and thus could not be routinely used. Recent technical breakthrough includes bacterial artificial chromosome based high efficiency gene targeting (Song et al., 2010), as well as the successful engineering of two systems of site-specific nucleases, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) (Hockemeyer et al., 2009; Hockemeyer et al., 2011). The two engineered nucleases are composed of programmable and sequence-specific DNA-binding modules, which bring the nucleases to specific genomic site to introduce a DNA double-strand break. However, these two technologies have several limitations, including the time-consuming and labor-intensive experimental design, and the risk of off-targeting mutations (Gaj et al., 2013). More recently, a new genome-editing technology, denoted the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, has been developed for efficient gene targeting in cells of various species, including zebrafish (Hwang et al., 2013), mouse (Wang et al., 2013), monkey (Niu et al., 2014), and human (Cong et al., 2013; Mali et al., 2013b). In this system, Cas9 nuclease is targeted to a specific genomic site by complexing with a guide RNA, which hybridizes a 20-nucleotide DNA sequence immediately preceding an NGG motif, introducing a double-strand break three nucleotides upstream of the NGG motif (**ek et al., 2012). Compared to ZFNs and TALENs, CRISPR/Cas9 system offers simple experimental design and very high targeting efficiency (Ding et al., Full size image

In this study, we demonstrate that Cas9n can induce precise gene targeting via HR in hESCs with a long DNA donor template. The targeting efficiency is around 5% (2/37) in HUES3 cells (Fig.1F). In contrast to the oligonucleotide-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 single-crossover clones with homologous recombination occurred only at the 5′ homologous arm. Therefore, our findings highlight the importance to confirm the homologous recombination events at both arms of the introduced donor DNA. In this context, in order to identify correctly targeted clones, both the upstream and downstream HR events as well as random integration of the template should be verified.

In summary, our studies indicate that Cas9n can achieve precise gene targeting via HR in hESCs with a long DNA template, allowing high fidelity and complex genetic manipulation of hESCs. One such application is to develop lineage-specific reporter lines to trace the lineage differentiation of hESCs.

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Footnotes

We thank Dr. Zhao Chen for helpful discussion. This work was supported by Grants from National Natural Science Foundation of China (Grant Nos. 81172828 and 81372494) and from California Institute for Regenerative Medicine (TR3-05559).

Zhili Rong, Shengyun Zhu, Yang Xu and Xuemei Fu declare that they have no conflict of interest. All hESC work in this study has been approved by the Institutional Embryonic Stem Cell Research Oversight Committee (ESCRO) of University of California, San Diego and Shenzhen Children’s Hospital.

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Shenzhen Children’s Hospital, 7019 Yitian Road, Shenzhen, 518026, China
Shengyun Zhu & Xuemei Fu
Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA, 92093, USA
Zhili Rong & Yang Xu
Chongqing Medical University, Chongqing, 400016, China
Xuemei Fu

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Zhili Rong
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Shengyun Zhu
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Yang Xu
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Xuemei Fu
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Correspondence to Xuemei Fu.

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Rong, Z., Zhu, S., Xu, Y. et al. Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template. Protein Cell 5, 258–260 (2014). https://doi.org/10.1007/s13238-014-0032-5

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Published: 14 March 2014
Issue Date: April 2014
DOI: https://doi.org/10.1007/s13238-014-0032-5

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Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template

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