Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system

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.

Highly Efficient Mouse Genome Editing by CRISPR Ribonucleoprotein Electroporation of Zygotes

“…1B), an essential enzyme for pigment synthesis (24). We generated the Tyr sgRNA using a cloning-free in vitro transcription method (supplemental Fig.…”

“…Derived from the bacterial type II CRISPR/Cas system, the Cas9 nuclease can be directed by a programmable single guide RNA (sgRNA) to induce double strand breaks at specific loci, triggering the NHEJ or HDR repair pathways to yield desired sequence modifications (18 -21). Using CRISPR, a variety of simple and sophisticated genomic modification schemes have been generated in mouse models, including precise sequence replacement (22)(23)(24), genome insertions/deletions (25,26), and chromosomal translocations (27). The standard practice for CRISPR editing in mice relies on microinjection of Cas9 DNA/mRNA and sgRNAs into one-cell zygotes (15,28), a process that remains laborious, costly, and low throughput with considerable technical barriers.…”

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

“…Mutations often appear in the embryo after the first cell divisions, such that most animals are mosaics. This is immediately observable for the tyrosinase gene as homozygous mutations in this gene cause albinism (Mizuno et al 2014). Mosaicism might result from the time taken for the construct to be transcribed and for Cas9 mRNA to be translated after pronucleus injection in the embryo.…”

CRISPR/Cas9: The Leading Edge of Genome Editing Technology

“…The gRNA and Cas9 can be expressed using either separate vectors or a single combined vector. pX330, a single vector expressing both gRNA and Cas9 nuclease with human U6 and chicken beta-actin hybrid (CBh) promoters, respectively, was originally developed by the Feng Zhang laboratory ) and has been very widely used for cell and animal genome editing (Mashiko et al 2013(Mashiko et al , 2014Ran et al 2013 ;Matsunaga and Yamashita 2014 ;Mizuno et al 2014 ;Park et al 2014 ;Yin et al 2014 ).…”