Efficient generation of large-scale genome-modified mice using gRNA and CAS9 endonuclease

Abstract

The generation of genome-modified animals is a powerful approach to analyze gene functions. The CAS9/guide RNA (gRNA) system is expected to become widely used for the efficient generation of genome-modified animals, but detailed studies on optimum conditions and availability are limited. In the present study, we attempted to generate large-scale genome-modified mice with an optimized CAS9/gRNA system, and confirmed the transmission of these mutations to the next generations. A comparison of different types of gRNA indicated that the target loci of almost all pups were modified successfully by the use of long-type gRNAs with CAS9. We showed that this system has much higher mutation efficiency and much lower off-target effect compared to zinc-finger nuclease. We propose that most of these off-target effects can be avoided by the careful control of CAS9 mRNA concentration and that the genome-modification efficiency depends rather on the gRNA concentration. Under optimized conditions, large-scale (~10 kb) genome-modified mice can be efficiently generated by modifying two loci on a single chromosome using two gRNAs at once in mouse zygotes. In addition, the normal transmission of these CAS9/gRNA-induced mutations to the next generation was confirmed. These results indicate that CAS9/gRNA system can become a highly effective tool for the generation of genome-modified animals.

Figure 1.

Comparison of structure and function between long and short gRNAs. (A) Schematic illustrations of long and short gRNAs for Rosa26 target locus. Target sequence was underlined. (B) Genome modification efficiency of long and short gRNAs evaluated by T7EI assay. Genomic PCR amplicons of blastocysts derived from fertilized eggs injected with CAS9 mRNA (100 µg/ml), and long or short gRNA (10 µg/ml) were subjected for T7EI assay. Experiments were repeated for six times. An example of typical results of T7EI assay was shown in upper part. The arrow and arrowhead indicate PCR amplicons and digested fragments by T7EI, respectively. Calculated efficiencies were shown as boxplot in lower part.

Figure 2.

Generation of Rosa26-targeted mice mediated by CAS9 and long-gRNA. (A) Schematic illustration of Rosa26 gene structure and sequences of wild-type and mutated alleles around the target locus. Arrows indicate the loci of PCR primers. The targeted locus of gRNA and PAM domain were indicated in the wild-type sequence by underline and bold letters, respectively. Examples of modified-allele sequences obtained from Rosa26 targeted pups are shown below in no particular order. Deleted and inserted nucleotides are indicated by hyphens and small letters, respectively. (B) Comparison of mutation efficiencies of Rosa26 target locus among various concentrations of CAS9 mRNA and gRNA injected into pronuclear-stage embryos. Numbers in the bars indicate the numbers of mutated pups/total pups. (C) Mutation efficiencies ofpotential off-target loci for Rosa26 target sequence. Mismatch nucleotides in examined sequences are shown by bold with underlines. N.D. indicates ‘not determined’. ‘a’ denotes concentrations of CAS9 mRNA + gRNA (µg/ml). ‘b’ denotes number of total pups. The locus information of these sequences are shown in Supplementary Figure S5.

Figure 3.

Comparison of toxicity and mutation efficiency between CAS9/gRNA system and ZFN on Cdkn1b locus. (A) Cdkn1b target sequences of gRNA and ZFNs. (B) Developmental competence of preimplantation embryos injected with CAS9/gRNA (100 + 10 µg/ml) or ZFNs (5 µg/ml each). Experiments were repeated three times, and the average blastocyst formation rates (mean + SD) are shown. (C) DNA-double-strand breaks of CAS9/gRNA-, ZFN- or non-injected zygotes were visualized by immunocytechemistry of γH2AX (upper panels) at 8 h after microinjection. Pronuclei visualized by propidium iodide (PI) staining (middle panels) and bright field of embryos (lower panels) are also shown. (D) The results of T7EI assay. Genomic PCR amplicons of blastocysts obtained from the previously mentioned experiments were subjected for T7EI assay. Arrow and arrowhead indicate PCR amplicons and digested fragments by T7EI, respectively. The percentage of digestion fragment was calculated and shown under each lane.

Figure 4.

Generation of double-mutated or large-scale deleted mice using two long gRNAs targeted Hprt exon1 and exon2. Different concentrations of CAS9 mRNA and two gRNAs were injected at once into pronuclear-stage embryos, and 1-cell or 2-cell stage embryos were transferred into oviducts. (A) Schematic illustration of Hprt gene structure and sequences around the target loci and obtained large-scale deleted alleles. Arrows in the schema indicate the loci of PCR primers, and the targeted sequences of gRNAs and PAM domain are indicated by underlines and bold letters, respectively. Sequences of all large-scale modified alleles, which were deleted ∼10 kb extended over the two target loci, are shown with respect to each concentration of CAS9 mRNA and gRNAs used. The hyphens and small letters denote deleted and inserted nucleotides, respectively. (B) Mutation efficiencies of Hprt loci. As target-locus of Hprt exon1 in all pups were mutated, the mutation efficiencies of exon2 are identical to the double mutated efficiencies. Results of different RNA concentrations were shown by black bars (100 μg/ml CAS9 and 10 + 10 μg/ml each gRNA), gray bars (10 μg/ml CAS9 and 10 + 10 μg/ml each gRNA) and white bars (10 μg/ml CAS9 and 1 + 1 μg/ml each gRNA). Numbers in the bars indicate the numbers of mutated pups/total pups. (C) An example of sequencing raw data from a pup with large-scale deletion.

Figure 5.

Transmission of mutations generated by CAS9/gRNA system. (A) Target sequences obtained form tail-tip DNA of F0 male and female mated, and F1 pups are shown with wild-type sequence. The sequences on the same line, which are connected by double slash, are obtained from same alleles. The hyphens and small letters denote deleted and inserted nucleotides, respectively. (B) The F0 female and obtained F1 pups used for the sequence analyses. (C) A pedigree obtained from analyses in (A). The identical alleles are shown by the same colors. The large-scale deletion of F0 male is wholly transmitted to the female pups. F0 female is genetically mosaic, and the target sequences of germ cells and tale-tip are different.