Generation of mutant mice via the CRISPR/Cas9 system using FokI-dCas9

Abstract

Genome editing, which introduces mutations in genes of interest using artificial DNA nucleases such as the ZFN, TALEN, and CRISPR/Cas9 systems in living cells, is a useful tool for generating mutant animals. Although CRISPR/Cas9 provides advantages over the two other systems, such as an easier vector construction and high efficiency of genome editing, it raises concerns of off-target effects when single guide RNA (gRNA) is used. Recently, FokI-dCas9 (fCas9), a fusion protein comprised of the inactivated mutant form of Cas9 and the DNA nuclease domain of FokI, has been developed. It enables genome editing with reduced risks of off-target effects in mammalian cultured cell lines, as fCas9 requires gRNAs to bind opposite strands with an appropriate distance between them. Here, we demonstrated that fCas9 efficiently generates living mutant mice through microinjection of its mRNA and gRNAs into zygotes. A comparison of the relative efficiencies of genome editing using fCas9 and other modified Cas9s showed that these mutagenesis efficiencies are similar when the targets of two gRNAs are separated by an appropriate distance, suggesting that in addition to the ease of vector construction, fCas9 exhibit high efficiency in producing mutant mice and in reducing risks of off-target effects.

Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

“…Specific sgRNA sequences were designed using the CRISPR Design Tool (http://genome-engineering.org/). sgRNAs for Irx3 , Irx5 , and Cas9 mRNA were prepared as previously described [20, 21]. Three sgRNA target sequences were cloned into sgRNA cloning vectors by inverse PCR using primers pairs Irx3_sg-F/Irx3_sg-R, Irx5_sg1-F/Irx5_sg1-R, and Irx5_sg2-F/Irx5_sg2-R (Table 1).…”

“…The fertilized eggs were collected in M2 medium. Microinjection was carried out as previously described [21]. Embryos were cultured and transferred to pseudopregnant ICR female mice at the two-cell stage.…”

Gene editing in mouse zygotes using the CRISPR/Cas9 system

“…Various modifications of the CRISPR/Cas9 system to reduce the off-target frequencies have then been developed, like Cas9 nickases [29,30], Cas9-FokI fusions [31], high-fidelity variants eCas9(1.1) [32] and Cas9-HF [33], truncated guide RNAs [34], orthologous or inducible Cas proteins [35][36][37][38]. Validation of such alternative nucleases in mouse zygotes has been reported for SpCas9 D10A nickase [39,40], AsCpf1 [41,42], LbCpf1 [42], St1Cas9 [43], Fok-dCas9 [40,44], and SaCas9 [45]. These improvements are very helpful for animal or cell culture models in which even rare off-target mutations are deleterious, like the generation of isogenic cell lines or in somatic gene therapy.…”

CRISPR/Cas9: a breakthrough in generating mouse models for endocrinologists

“…Another interesting attempt to limit off-target cleavages is the use of the Cas9-Fok1 fusion protein, in which the nuclease activity of Cas9 is eliminated and replaced by the nuclease activity of Fok1, which can only cleave DNA as a homodimer. Therefore, Cas9-Fok1 cuts only if two sgRNAs target neighboring genomic sequences (Hara et al 2015). Both strategies require that two PAM sequences are found at the appropriate distance on opposite strands in the targeted locus.…”