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Review
. 2016 Jul 18;37(4):205-13.
doi: 10.13918/j.issn.2095-8137.2016.4.205.

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

Li-Fang Jin  1 , Jin-Song Li  2
Affiliations
Review

Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

Li-Fang Jin et al. Dongwuxue Yanjiu. .

Abstract

With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches.

Keywords: CRISPR/Cas9; Genetic modification; Haploid embryonic stem cells; Mouse.

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Figures

SgRNA guides the sgRNA/Cas9 complex to the target DNA. Cas9 then cleaves chromosomal DNA, resulting in site-specific DNA double-strand breaks (DSBs). Specificity is determined by an sgRNA-DNA hybrid and protospacer adjacent motif (PAM).
Figure 1
Structure and mechanism of sgRNA/Cas9
In vitro prepared Cas9 mRNA, sgRNA, and targeting template are microinjected into zygotes from super-ovulated females. The CRISPR/Cas9 system changes targeted DNA in embryos by HR or NHEJ. Developed embryos derived from injected zygotes are implanted into pseudo-pregnant mice to produce a mutant F0 generation.
Figure 2
Germline-modified mouse from embryo editing using the CRISPR/Cas9 system
DKO-AG-haESCs are derived from androgenetic haploid ESCs (AG-haESCs), in which differentially methylated regions (DMRs) of H19 and Gtl2 are deleted. DKO-AG-haESCs with multiple-gene modifications, including constitutive expression of Cas9 and sgRNA library, transient expression of Cas9 and sgRNA library, or multiple-gene knockout or knockin, generate semi-cloned mice with multiple-gene modifications for genetic screening using intracytoplasmic AG-haESC injection (ICAHCI) technology.
Figure 3
H19 and Gtl2 double-knockout androgenetic haploid ESCs (DKO-AG-haESCs) carrying multiple-gene modifications support the efficient generation of different mutant mice in one step (Zhong et al., 2015)
See this image and copyright information in PMC

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