Using CRISPR/Cas9 Technology To Establish An In Situ Knock-in Mouse Model Of Huntington's Disease (HD)

Background:Huntington's disease?HD?is a neurodegenerative autosomal dominant genetic disease.The most obvious clinical symptom is involuntary dance-like movement of the patient,so Huntington's disease is also called Huntington's chorea.Studies have shown that HD has an age-dependent feature,that is,with the increase of age,the condition of HD patients will gradually become more serious until death.It is currently believed that the cause of human HD is that the abnormal expansion of the CAG sequence occurs in the Huntington gene?Htt?exon 1 on the number 4 autosome,resulting in misfolding of the normal huntingtin protein,which forms the mutant huntingtin protein?mHtt?.The cells can not completely remove mHtt.When mHtt accumulates to a certain extent,it causes degeneration of brain striatum neuron cells and atrophic necrosis of brain tissue.In general,the longer length of the CAG abnormal mutation,the earlier onset age of HD,and the more serious disease phenotype.There are three main HD mouse models commonly used:1)the"knock-out mouse model"that targets gene knockout to destroy pathogenic genes;2)the"transgenic mouse model"that conditionally overexpresses the N-terminus of the mHtt or the full-length mutant protein by introducing a exogenous gene or promoter;3)the"knock-in mouse model"that changes the endogenous gene only under the control of the mouse's own endogenous promoter.At present,with the rapid development of emerging genetic editing technologies such as CRISPR/Cas9,researchers have more options in constructing different HD models.It provides a new platform for drug screening and new drug development.Objective:An HD in situ knock-in mouse model was constructed by using CRISPR/Cas9 technology to knock 150Q into exon 1 of mouse Htt gene without adding exogenous gene?or exogenous promoter?to change specific endogenous genes.Methods:?1?According to the C57BL/6J mouse Htt gene information included in NCBI,the Cas9/gRNA target sequences were designed.And the digestion activity of Cas9/gRNA was detected by preparing transcribed gRNA and digested DNA in vitro.The L-terminal and R-terminal gRNA targets with better activity were selected for pairing.?2?The 150Q Donor plasmid was designed by reference to the Htt gene information and the active target position of the gRNA sequences.?3?Cas9 mRNA,gRNA,150Q Donor DNA and other components had been microinjected into the zygotes,and then the embryos were transplanted into the recipient females to be produced.?4?F0 generation mice were identified for their genotype after birth,and F0heterozygous mice were selected and mated with wild-type mice to obtain F1generation mice.After genotype identification of F1 generation,F1 heterozygous mice were mated with wild-type mice to obtain F2 generation mice.Then,the F2heterozygous mice with the correct genotype were mated with each other,and it was hoped that homozygotes would be born in the F3 generation mice.?5?RNA had been respectively extracted from brain,cerebellum,brainstem,liver,kidney,lung,heart,spleen of HD^150Q/7Q mice at 9 months,5 months,and 2 months and HD^7Q/7Q mice from the same litter.It had been detected whether RNA had 150Q?or 7Q?transcription.?6?Proteins had been respectively extracted from brain,cerebellum,brainstem,liver,kidney,lung,heart,spleen of HD^150Q/7Q mice at 9 months,5 months,and 2 months and HD^7Q/7Q mice from the same litter.Three protein antibodies,Htt protein?MAB2166,Millipore?,mutant Htt protein?MAB1574,Millipore?,and?-actin?13E5,Cell Signaling Technology?were tested.Results:?1?A total of twenty gRNA target sequences were designed.After detecting Cas9 digestion activity in vitro,the Htt-L9 and Htt-R9 with higher activity were selected to construct No.9 gRNA target sequences.?2?After microinjections and embryos transfer,six F0 mice were successfully born.Two of six mice were correct genotypes,and they were heterozygous males.?3?As of November 17,2018,filial generation?F1,F2,F3?were born 118 mice,of which 38 males and 31 females had correct genotypes.All of them were heterozygous.No homozygous mice.?4?Because the 150Q model mice were heterozygous(HD^150Q/7Q),the RNA of each tissue in the HD^150Q/7Q mice had 150Q and 7Q simultaneous transcription,while the normal mice(HD^7Q/7Q)had only 7Q transcription.?5?In the case of consistent expression levels of?-actin,the expression of Htt protein?MAB2166,Millipore?was expressed in all tissues of HD^150Q/7Q mice and HD^7Q/7Q mice,but mutant Htt protein?MAB1574,Millipore?was only detected in the tissues of HD^150Q/7Q mice.Conclusion:CRISPR/Cas9 technology and homologous recombination method were used to replace 7Q with 150Q.The results of mice genomic DNA identification,RNA transcription and related protein expression showed that the construction of the HD model of 150Q in situ knock-in mouse was completed,and heterozygous mice(HD^150Q/7Q)were successfully obtained.HD may have late-onset effect,and our in situ knock-in model mice are heterozygous,so 150Q mice have not yet shown significant disease characterization of HD.In addition,the body weight and activity analysis were tested,but no obvious difference was found between heterozygous mice(HD^150Q/7Q)and normal mice(HD^7Q/7Q).