Generation of chimeric mice with spermatozoa fully derived from embryonic stem cells using a triple-target CRISPR method forNanos3†

Abstract

Conditional knockout (cKO) mice have contributed greatly to understanding the tissue- or stage-specific functions of genes in vivo. However, the current cKO method requires considerable time and effort because of the need to generate two gene-modified mouse strains (Cre transgenic and loxP knockin) for crossing. Here we examined whether we could analyze the germ cell-related functions of embryonic lethal genes in F0 chimeric mice by restricting the origin of germ cells to mutant embryonic stem cells (ESCs). We confirmed that the full ESC origin of spermatozoa in fertile chimeric mice was achieved by the CRISPR/Cas9 system using three guide RNAs targeting Nanos3, which induced germ cell depletion in the host blastocyst-derived tissues. Among these fertile chimeric mice, those from male ESCs with a Dnmt3b mutation, which normally causes embryo death, also produced F1 mice derived exclusively from the mutant ESCs. Thus, our new chimeric strategy readily revealed that Dnmt3b is dispensable for male germ cell development, in agreement with a previous cKO study. Our new approach enables us to analyze the germ cell functions of embryonic lethal genes in the F0 generation without using the current cKO method.

Electroporation-Mediated Genome Editing of Livestock Zygotes

“…It may be that cloning ESCs increases the efficiency of cloning success relative to SCNT (McLean et al, 2020). Alternatively, embryo complementation or injecting donor totipotent edited stem cells into genome edited knockout, germline ablated host embryos (Ciccarelli et al, 2020;Miura et al, 2020), or edited primordial germ cells in the case of poultry (Woodcock et al, 2019), may provide an alternative approach to produce animals that transmit gametes derived solely from an edited cell line. This could help to resolve the problem of mosaicism that is frequently associated with electroporation-mediated genome editing of mammalian zygotes.…”

Progress of genome editing technology and developmental biology useful for radiation research

“…The pups born from the resulting zygotes contain biallelic mutations at nearly 100% efficiency. Since screening and analysis of the gene function can be achieved without establishing or maintaining multi-generated KO animal strains, this method has already been applied to KO of various genes [ 59 , 61 , 62 , 64 , 65 ] and mouse strains [ 63 ].…”

“…Furthermore, a new system combining the triple-target CRISPR system with a blastocyst complementation method has recently been developed [ 63 ]. In the blastocyst complementation method, a target organ exclusively derived from embryonic stem cells (ESCs) or induced pluripotent cells (iPSCs) can be generated in chimeric animals by injecting these cells into blastocysts in which the development of the original target organ is prevented [ 81 , 82 ].…”

“…The concept of the blastocyst complementation/triple-target CRISPR-combined method was validated in germ cells, whose loss was caused by KO of the gene coding nanos C2HC-type zinc finger 3 ( Nanos3 ) [ 87 ]. The germ loss phenotype was confirmed in several mouse strains by targeting Nanos3 in the triple-target CRISPR method [ 63 ]. Mutating the DNA methyltransferase 3B ( Dnmt3b )-coding gene was reported to causes embryonic death [ 88 ], though the dispensable role of Dnmt3b in germ cell development was demonstrated by a germ cell-specific cKO study [ 89 ].…”

“…From fertile chimeric male mice generated by injecting Dnmt3b −/− male ESCs into blastocyst carrying biallelic mutations of Nanos3 , only pups with ESC-derived coat color were born. Since the contribution of pluripotent stem cells to germ cells of F0 chimeric mice can be evaluated by the coat color of F1 offspring, it was suggested that spermatozoa of the fertile chimeras were fully derived from the ESCs ( Figs 4A and B ) [ 63 ].…”