Targeted Germline Modifications in Rats Using CRISPR/Cas9 and Spermatogonial Stem Cells

Abstract

SUMMARY Organisms with targeted genomic modifications are efficiently produced by gene editing in embryos using CRISPR/Cas9 RNA-guided DNA endonuclease. Here, to facilitate germline editing in rats, we used CRISPR/Cas9 to catalyze targeted genomic mutations in rat spermatogonial stem cell cultures. CRISPR/Cas9-modified spermatogonia regenerated spermatogenesis and displayed long-term sperm forming potential following transplantation into rat testes. Targeted germline mutations in Epsti1 and Erbb3 were vertically transmitted from recipients to exclusively generate “pure”, non-mosaic mutant progeny. Epsti1 mutant rats were produced with or without genetically selecting donor spermatogonia. Monoclonal enrichment of Erbb3-null germlines unmasked recessive spermatogenesis defects in culture that were buffered in recipients, yielding mutant progeny isogenic at targeted alleles. Thus, spermatogonial gene editing with CRISPR/Cas9 provided a platform to generate targeted germline mutations in rats, and to study spermatogenesis.

Leapfrogging: primordial germ cell transplantation permits recovery of CRISPR/Cas9-induced mutations in essential genes

“…Several potential solutions to this problem have been published. In rodents, the introduction of programmable nucleases, performed either in vivo or in vitro, into adult spermatogonial or oogonial stem cells has been accomplished (Fanslow et al, 2014;Chapman et al, 2015;Sato et al, 2015;Takahashi et al, 2015;Wu et al, 2015). These mutated germ cell precursors variably contribute to the germline while endogenous unmutated germ cells remain.…”

Modeling Disease In Vivo With CRISPR/Cas9

“…Targeted genomic manipulation in the rat was transformed by the advent of ZFN and TALEN technology, which provided a means to create mutant strains without the need for ESC cultures. And, though ZFNs and TALENs have proven effective, the field has been quick to adapt the newest CRISPR tools [75–79] to produce a range of germline mutant strains, as well as knock-in fluorescent reporters and Cre and LoxP conditional mutants that have proven so effective in the mouse [80]. As is already clear in cell culture models, the simplicity of CRISPR will likely see it become the dominant genome-engineering tool in the rat, and this will translate to an expanded array of rat disease models [81].…”

Germline genome-editing research and its socioethical implications

“…Moreover, germline gene modifications are also attainable via genome editing of spermatogonial stem cells (SSCs), potentially preventing mosaicism in offspring by avoiding the totipotent states that accompany embryogenesis. Successful editing using the SSC approach has been demonstrated in mice [38] and in rats [39]. Of note, Reddy et al demonstrated that mitochondria-targeted TALENs (mito-TALENs) can be used in mouse oocytes fused with cells from a patient to eliminate human mitochondrial DNA (mtDNA) mutations [40].…”