Abstract
Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.
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Acknowledgments
This work was supported by Grants-in-Aid for Scientific Research (S) (to S.T., F.S., and K.Y.) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan. We thank the members of the Yagami Laboratory for helpful discussions and encouragement.
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Supplementary Fig. 1
C57BL/6J mice with albino and mosaic phenotype. Completely white (26 of the albino founders)/black (5 of the black mice) coats and mosaic mice with black and white coats (11 of the mosaic founders) were in one-week-old founders. (PDF 9101 kb)
Supplementary Fig. 1
Genomome sequences in albino founders without Tyr G291T mutation. The red letters indicate the CRISPR target site. The large letter “G” in red is the target single nucleotide. The green and blue letters indicate deletion and insertion sequences in albino mice, respectively. (PDF 11770 kb)
Supplementary Fig. 3
Sequences of off-target candidate. Each off-target candidate was compared by the reference sequence targeted. The red boxes mean the different nucleotides between candidate and reference sequences. (PDF 111 kb)
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Mizuno, S., Dinh, T.T.H., Kato, K. et al. Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system. Mamm Genome 25, 327–334 (2014). https://doi.org/10.1007/s00335-014-9524-0
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DOI: https://doi.org/10.1007/s00335-014-9524-0