The Regulation Mechanism Of Tyrosinase(TYR) Geneon Hair Color

Tyrosinase was a kind of aerobic enzyme with copper,and also known as polyphenol oxidase,it was widely existed in microbes,plants and animals.It had a variety of characteristic catalytic activities and physiological functions,and had received extensive attentions.In recent years,the amount of fur animals in our country is enormous,domestic and international market of fur quality requirements more and more high,the hair color has become an important factor in determining the quality of fur,Therefore,it is of great significance to master the genetic mechanism of fur color and the factors that affect the quality of fur.The main function of tyrosinase is to promote the formation of melanin.Tyrosinase gene mutation can lead to albinism.The overall incidence of albinism in Chinese population is 1 to 18000.Therefore,it is very important to clarify the pathogenesis of tyrosinase for albinism related treatment.The CRISPR RNA-guided Cas9 nuclease genetargetingsystem has been extensively used to edit the genome of several organisms.In this study,A series of experiments on the TYR gene in rabbits via a dual sg RNA directed CRISPR/Cas9 system,including the whole TYR gene 105 Kb fragment knockout,3'UTR region knockout and point mutation.The results showed that TYR large fragment knockout rabbits had color whitening symptoms.The same genetypes and symptoms were found in the F1 generation rabbits,suggesting that the TYR gene can directly affect the formation of melanin,resulting in albinism occurs,and that large-fragment deletions can beefficiently transmitted to the germline and stably inherited in offspring.In addition,m RNA 3'UTRs are thought to play an important role in RNA replication and protein translation.However,there is no relevant animal experiment at present.Therefore,we used the CRISPR /Cas9 system to control the 3'UTR region of the TYR gene to observe the change in coat color.As expected,gray coat color and reduced melanin in hair follicles and irises was found in the mutated rabbit.The graying phenotype was also found in the F1 generation,indicating that the deletion of the 3'UTRs of the TYR gene resulted in TYR gene expression decreased,lead to less melanin produced.Thus,we provide the first evidence that 3'UTR region plays an important role in the regulation of TYR gene expression and animal coat color Additionally,CRISPR/Cas9-mediated fragment deletions can facilitate genotype to phenotype studies of UTRs or non-coding RNAs in future.Finally,we successfully obtained TYR gene knock in rabbit use the CRISPR/Cas9 system,the hair and the iris color is black,and albinism symptom has been significantly improved,which can be stably inherited in F1 generation.Through in vitro experiments,we found that the mutation of TYR gene resulted in the failure of the copper binding site of the enzyme center,resulting in the decrease of tyrosinase activity and the inhibition of melanin formation,lead to skin whitening.It also shows that CRISPR/Cas9 system can be used effectively in gene therapy and other related studies in animals.In summary,tyrosinase expression will affect the coat color changes and melanin production.It is also proved that CRISPR/Cas9 system can efficiently perform whole gene deletion,UTR region,non coding RNA region knockout and point mutation in animals.