Improved efficiency of genome editing by constitutive expression of Cas9 endonuclease in genetically-modified mice

Abstract

Despite its convenience and precision, CRISPR-based gene editing approaches still suffer from off-target effects and low efficiencies, which are partially rooted in Cas9, the nuclease component of the CRISPR/Cas9 system. In this study, we showed how mouse genome editing efficiency can be improved by constitutive and inheritable expression of Cas9 nuclease. For this goal, a transgenic mouse line expressing the Cas9 protein (Cas9-mouse) was generated. For in vitro assessment of gene editing efficiency, the Cas9-mice were crossed with the EGFP-mice to obtain mouse embryonic fibroblasts (MEF) expressing both EGFP and Cas9 (MEF_Cas9-EGFP). Transfection of these cells with in vitro transcribed (IVT) EGFP sgRNA or phU6-EGFP_sgRNA plasmid led to robust decrease of Mean Fluorescent Intensity (MFI) to 8500 ± 1025 a.u. and 13,200 ± 1006 a.u. respectively. However, in the control group, in which the MEF_EGFP cells were transfected with a pX330-EGFP_sgRNA plasmid, the measured MFI was 16,800 ± 2254 a.u. For in vivo assessment, the Cas9-zygotes at two pronuclei stage (2PN) were microinjected with a phU6-Hhex_sgRNA vector and the gene mutation efficiency was compared with the wild-type (WT) zygotes microinjected with a pX330-Hhex_sgRNA plasmid. The analysis of born mice showed that while the injection of Cas9-zygotes resulted in 43.75% Hhex gene mutated mice, it was just 15.79% for the WT zygotes. In conclusion, the inheritable and constitutive expression of Cas9 in mice provides an efficient platform for gene editing, which can facilitate the production of genetically-modified cells and animals.

Keywords: CRISPR-Cas9; Gene knock-out; Genetically-modified cells and mice; Microinjection.

Fig. 1

Construction of INPGK-CAS9 vector from pX330 and INPGK vectors. The Cas9 encoding sequence was obtained from a pX330 plasmid through EcoRI and AgeI restriction enzymes digestion and subsequently replaced the EGFP gene in a INPGK plasmid, which served as the backbone vector. The resulting vector, INPGK-CAS9, was incorporated into mice genome to generate the Cas9 transgenic mice

Fig. 2

a Schematic representation of Cas9 transgenic mouse generation. The Cas9-expressing cassette along with the flanked insulators (which appeared as a 7230 bp fragment on the gel electrophoresis) was injected into 2PN stage zygotes, and then the zygotes implanted into a surrogate mouse until the breeding of offspring. b Western blot confirmed the Cas9 expression. Each well represents samples of various body organs derived from either Cas9 or WT mice. Lanes 1–6 belonging to Cas9 mouse’s kidney, liver, testis, bone marrow, hearts, and brain, respectively, and lanes 7–9 being derived from WT mouse’s kidney, liver, and testis. Beta actin was used as loading control

Fig. 3

CRISPR/Cas9-mediated EGFP knocking out in MEF cells. a The figure represents schematic strategy for in vitro EGFP gene knockout. As displayed, the Cas9-mice were crossed with the EGFP mice and the MEFCas9-EGFP cells were isolated from 12.5 days old embryos and cultured. The IVT EGFPsgRNA and phU6-EGFPsgRNA plasmid were transfected separately into the MEFCas9-EGFP cells. The pX330-EGFPsgRNA vector was also transfected into the MEFEGFP cells which served as the control of this study. b The diagram represents the measured MFI in three MEF populations using flow cytometry. As shown, the level of MFI in the different groups of MEFs was measured as IVT EGFPsgRNA < phU6-EGFPsgRNA < pX330-EGFPsgRNA

Fig. 4

a. Microinjection-based strategy for in vivo gene knockout. The phU6-HhexsgRNA vector was microinjected into the Cas9 2PN zygotes. In parallel, the pX330-HhexsgRNA was injected into the WT embryos. The offspring were then assessed for the extent of genetic alterations in the Hhex locus. b Mutation positions throughout Hhex alleles in WT and Cas9 embryos’ offspring. Three out of 19 pups, which had been injected with the pX330-HhexsgRNA into the WT zygotes, carried mutant Hhex allele; while seven out of 16 pups, which had been injected by the phU6-HhexsgRNA into the Cas9-zygotes displayed indels at sgRNA targeting site