Efficient generation of cloned cats with altered coat colour by editing of the KIT gene.

Coat colour largely determines the market demand for several cat breeds. The KIT proto-oncogene (KIT) gene is a key gene controlling melanoblast differentiation and melanogenesis. KIT mutations usually cause varied changes in coat colour in mammalian species. In this study, we used a pair of single-guide RNAs (sgRNAs) to delete exon 17 of KIT in somatic cells isolated from two different Chinese Li Hua feline foetuses. Edited cells were used as donor nuclei for somatic cell nuclear transfer (SCNT) to generate cloned embryos presenting an average cleavage rate exceeding 85%, and an average blastocyst formation rate exceeding 9.5%. 131 cloned embryos were transplanted into four surrogates, and all surrogates carried their pregnancies to term, and delivered 4.58% (6/131) alive cloned kittens, with 1.53% (2/131) being KIT-edited heterozygotes (KITD17/+). The KITD17/+ cats presented an obvious darkness reduction in the mackerel tabby coat. Immunohistochemical analysis (IHC) of skin tissues indicated impaired proliferation and differentiation of melanoblasts caused by the lack of exon17 in feline KIT. To our knowledge, this is the first report on coat colour modification of cats through gene editing. The findings could facilitate further understanding of the regulatory role of KIT on feline coat colour and provide a basis for the breeding of cats with commercially desired coat colour.

Bone morphogenetic protein 15 gene disruption affects the in vitro maturation of porcine oocytes by impairing spindle assembly and organelle function.

Bone morphogenetic protein 15 (BMP15) plays a crucial role in the porcine follicular development. However, its exact functions in the in vitro maturation (IVM) of porcine oocytes remain largely unknown. Here, through cytoplasmic injection of a preassembled crRNA-tracrRNA-Cas9 ribonucleoprotein complex, we achieved BMP15 disruption in approximately 54 % of the cultured porcine oocytes. Editing BMP15 impaired the IVM of porcine oocytes, as indicated by the significantly increased abnormal spindle assembly and reduced first polar body (PB1) extrusion. The editing also impaired cytoplasmic maturation of porcine oocytes, as reflected by reduced abundant of Golgi apparatus and impaired functions of mitochondria. The impaired IVM of porcine oocytes by editing BMP15 possibly was associated with the attenuated SMAD1/5 and EGFR-ERK1/2 signaling in the cumulus granulosa cells (CGCs) and the inhibited MOS/ERK1/2 signaling in oocytes. The attenuated MOS/ERK1/2 signaling may contribute to the inactivation of maturation promoting factor (MPF) and the increased abnormal spindle assembly, leading to reduced PB1 extrusion. It also may contribute to reduced Golgi apparatus formation, and impaired functions of mitochondria. These findings expand our understanding of the regulatory role of BMP15 in the IVM of porcine oocytes and provide a basis for manipulation of porcine reproductive performance.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP.

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/µL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/µL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.