Homologous recombination in human embryonic stem cells using CRISPR/Cas9 nickase and a long DNA donor template
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KeywordsBacterial Artificial Chromosome Human Embryonic Stem Cell Cluster Regularly Interspaced Short Palindromic Repeat Random Integration Cas9 Nuclease
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 (Jinek et al., 2012). Compared to ZFNs and TALENs, CRISPR/Cas9 system offers simple experimental design and very high targeting efficiency (Ding et al., 2013). However, some studies have also raised the concern about the off-target mutation effect of this system (Hsu et al., 2013; Mali et al., 2013a). To reduce the off-target mutations, a D10A mutant nickase version of Cas9 (Cas9n) has been developed to replace wild type Cas9 and shown to increase the ratio of homology-directed repair (HDR) to nonhomologous end joining (NHEJ) (Cong et al., 2013; Mali et al., 2013b). However, the targeting efficiency of Cas9n is much lower than wild type Cas9, raising the concern that it cannot be applied in homologous recombination (HR) in hESCs.
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.
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.
- Niu Y, Shen B, Cui Y, Chen Y, Wang J et al (2014) Cell. doi: 10.1016/j.cell.2014.01.027
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