A Novel Coat Color Of Rabbits Generated Via MC1R Mutation With CRISPR/Cas9 System

The coat color trait is one of the important economic traits of livestock.The MC1R gene is an important gene involved in the formation of coat color and produces different coat colors by regulating the melanogenesis.Rabbits are important economic animals in modern animal husbandry and excellent model animals in biological research and translational medicine.In this study,the MC1R knockout rabbit with novel pale-yellow coat color was successfully produced using the CRISPR/Cas9 gene editing system.The MC1R knockout rabbit can be used as a MC1R knockout model for MC1R function research,and it provided guidance for breeding animals with novel coat color by using CRISPR/Cas9.The main findings of this study are as follows:1.Cloning and analysis of rabbit MC1R gene coding sequence(CDS).In this study,MC1R gene CDS in Lianshan black rabbit,Fujian yellow rabbit,New Zealand white rabbit,giant gray rabbit and giant checkered rabbit were amplified by PCR.The amplified rabbit MC1R gene coding sequences were correct by sequencing and aligning the Gen Bank MC1R sequence.Bioinformatics analysis was performed on MC1R gene CDS.The multiple sequence alignment showed that there was no deletion mutation in the giant gray rabbit,and its allele belonged to wild type E.Lianshan black rabbit,New Zealand white rabbit and checkered Giant Rabbit had c.281?286del deletion,their alleles belonged to the black dominant type E~D;the Fujian yellow rabbit had the c.304?333del deletion,and the allele belonged to the recessive wild type e.2.Efficiency detection of CRSIPR/Cas9-mediated MC1R gene knockoutTwo sg RNAs target sequences were designed on the second and fifth transmembrane domains,and the CRISPR/Cas9 expression vectors were constructed.The targeting activity of the two sg RNAs were detected using the RGS fluorescence reporter system in 293T cell lines.The flow cytometry results showed that the targeting efficiencies of the two sg RNA pairs were 65.3%and76.1%,respectively.The plasmids expressing the two sg RNAs and Cas9 were co-transfected into rabbit embryonic fibroblasts by electroporation,and two sg RNAs were confirmed to cause long deletion of the MC1R gene by PCR and sequencing.The knockout efficiencies of simultaneous injection of two sg RNAs and injection of a single sg RNA were detected in rabbit fertilized eggs.PCR and T7E1 digestion assay showed that the knockout efficiency of simultaneous injection of two sg RNAs in fertilized eggs was 85.7%.The above results demonstrated the large fragment deletion of the rabbit MC1R gene can be efficiently produced by co-injecting two sg RNAs into the fertilized eggs.3.The production of MC1R knockout rabbitIn this study,MC1R knockout rabbits were produced by co-injection of two sg RNAs.After transplanting 58 fertilized eggs of Lianshan black rabbit with two sg RNA and Cas9 m RNA into 4 receptor rabbits,and eventually gave birth to 9offspring.PCR and sequencing confirmed that two(No.Y31 and Y32)of the above offspring were MC1R knockout positive rabbits.Y32 contained 4 MC1R gene editing types,and Y31 contained 2 gene editing types and wild type e allele.Both Y31 and Y32 showed novel pale-yellow coat color,but the color of Y31 was slightly darker than Y32.H&E staining analysis showed that the content of eumelanin in MC1R knockout rabbits hair follicles were significantly lower than that in wild type black rabbits.The protein structure prediction results indicated that various gene editing types in MC1R knockout rabbits can cause long fragment deletion or frameshift mutation to cause the stop codon to appear prematurely,resulting in truncation of MC1R protein and serious structure damage.The off-target test results showed that there was no off-target among the 16 potential off-target sites.The results of real-time quantitative PCR showed that the expression levels of TYRP1 and DCT genes,which regulate eumelanin synthesis,in MC1R knockout rabbits were significantly decreased compared with wild type yellow rabbits.These results showed that simultaneous injection of two sg RNA and Cas9m RNA into the fertilized eggs of Lianshan black rabbit can efficiently delete long fragments of the MC1R gene,which lead to a decrease in the expression of TYRP1and DCT genes that regulate the synthesis of true melanin,resulting in the novel pale-yellow coat color.