The CRISPR/Cas9 System-mediated Gene Knock-in Was Used To Construct A Mouse Model Of Hepatolenticular Degeneration Of Type R778L

Hepatolenticular degeneration is a rare autosomal recessive copper metabolic disorder,and the causative gene is ATP7B.Due to the base mutation,insertion or deletion of ATP7B gene,the function of ATP7B protein is impaired or even inactivated,which leads to deposition of copper ions in the body and induces liver damage and nervous system damage.In Asia,the most common type is R778L,but the pathogenesis of this type of mutation is still unclear.It is very necessary to establish an animal model for R778L.The third-generation gene editing technology represented by CRISPR/Cas9 has been widely used in various life research.One of the important applications is the construction of disease models.CRISPR/Cas9-mediated gene knockdown provides the possibility for us to construct a mouse model that targets the R778L mutation.Firstly,we compared the sequences of the ATP7B gene and protein in humans and mice by BLAST and found that both are highly conserved.This provides an evolutionary perspective for the theory of modeling hepatolenticular degeneration using mice as model animals.Among them,the human R778L mutation corresponds to the mouse R780L mutation.Secondly,we performed microinjection of mouse fertilized eggs through the CRISPR/Cas9 system and succeeded in obtaining mice carrying the point mutation.After mating and purifying the background,we obtained homozygous R780L mutant mice.Next,we examined whether R780L mice could mimic the symptoms of hepatolenticular degeneration,both from a pathological and physiological point of view.The experimental results show that R780L mice have significant copper ion deposition in the liver and elevated serum ALT and AST.Finally,we detected potential off-target sites through the Deep sequence and found no detectable off-target effects.Taken together,we successfully constructed a R780L mutant mouse aimed for human R778L mutation using CRISPR/Cas9-mediated gene knock-in.This provides a suitable vector for the elucidation of the exact pathogenesis of R778L hepatolenticular degeneration and gene therapy.Of course,our research is still in the preliminary stage,and the specific molecular mechanism of R778L has not been elucidated.It is worth mentioning that the animal model previously used for hepatolenticular degeneration is mostly a knockout model,which is inappropriate for highly heterogeneous diseases such as hepatolenticular degeneration.And we expect that,after our work,more accurate animal models for specific mutation types are constructed in order to identify the poorly correlation between genotype and phenotype in hepatolenticular degeneration.