| The CRISPR/Cas9 system has the advantages of speed,efficiency,and convenience,and has been widely used in research fields such as gene function,gene therapy,genetic engineering,animal model construction and functional gene screening.The system consists of Cas9 protein,crRNA(crispr RNA,crRNA)and tracrRNA(trans activating CRISPR RNA,tracrRNA).Cas9 performs the function of endonuclease in the presence of single stranded guided RNA(sgRNA),cutting the double strands of DNA at the target site and forming double strand breaks(DSBs).DSB can activate intracellular repair mechanisms to guide broken DNA towards non homologous end joining(NHEJ)or homologous recombination(HR).NHEJ is a highly efficient but error-prone pathway for repair,often causing insertion or deletion at the DSB,leading to frameshift mutations.HR is a precise repair pathway,but its efficiency is very low,and is different from NHEJ’s active occurrence throughout the cell cycle,HR is only active at the S and G2/M stages.Using DNA cleavage and recombination,combined with CRISPR/Cas9 gene editing technology and single stranded oligonucleotides(ssODN)is one of the commonly used gene mutation methods.This method can also be used to establish a point mutation mouse model.Both sides of the ssODN are homologous arms,and the middle contains the sequence to be knocked in.Although the addition of ssODN can guide repair to homology directed repair(HDR),how to improve the efficiency of HDR remains a problem that needs to be tackled.In order to improve the efficiency of recombination,we added chain exchange protein Rad51 during injection into mouse embryos and conducted a series of studies on the establishment of neural system disease models.The main research content and results are divided into the following three parts:The first part:To study the efficiency of knock-in mutations in the combination of sgRNA,Cas9 protein,ssODN,and Rad51 on sex chromosomes and autosomes,respectively.Using the gRNA design website,158 cut sgRNA was designed for the methylCpG binding protein 2(MeCP2)located on the sex chromosome,and tau-V337M sgRNA was designed for the microtubule associated protein gene(Mapt)located on the autosomal chromosome.The two sgRNAs were obtained through in vitro transcription.The 158 cut sgRNA was mixed with spCas9 protein and injected into the mouse fertilized egg,After sequencing embryos that have developed to blastocysts,it was found that 158 cut sgRNA has a high cutting efficiency in mouse embryos;After mixing tau-V337M sgRNA with spCas9 protein and injecting it into mouse fertilized eggs,it was found that tau-V337M sgRNA also has a high cutting efficiency in mouse embryos after sequencing embryos that have developed to blastocysts.Next,we injected 158 cut sgRNA,spCas9 protein,ssODN,and Rad51 into mouse fertilized eggs in different combinations to compare the difference in knock-in(KIT>C)efficiency at the target site.The results showed that the 158 cut sgRNA+Cas9 proteome had the most types of InDel(insertion and deletion)and the most complex cutting patterns.The main pattern was in situ insertion,followed by in situ delete,with both ectopic insertion and ectopic deletion;Compared with the 158 cut sgRNA+Cas9 protein group,the 158 cut sgRNA+Cas9 protein+Rad51 group also had in situ inserts,followed by in situ deletes,and also had ectopic deletes,but the type of InDel s in the 158 cut sgRNA+Cas9 protein+Rad51 group decreased by nearly half;Compared with the 158 cut sgRNA+Cas9 protein group,the 158 cut sgRNA+Cas9 protein+ssODN group had a decrease of nearly half in the types of InDel,and only in situ insert and in situ delete occurred;Compared with the 158 cut sgRNA+Cas9 protein group,the 158 cut sgRNA+Cas9 protein+Rad51+ssODN group had a decrease in the types of InDels,but the number of decreases was not significant.In situ inserts were the dominant,followed by in situ delete,and ectopic inserts were also present.Although the addition of Rad51 did not significantly reduce the type of InDel,the 158 cut sgRNA+Cas9 protein+Rad51+ssODN group was the only one among the four groups that experienced precise point mutations(KIT>C).Tau-V337M sgRNA,spCas9 protein,ssODN,and Rad51 were injected into mouse embryos in different combinations,and the differences in the knock-in(KIA>G)efficiency of different combinations at the target site were also compared.The results showed that the tau-V337M sgRNA+Cas9 proteome had the least types of InDel;Compared with the tau-V337M sgRNA+Cas9 protein group,the type of InDel in the tauV337M sgRNA+Cas9 protein+Rad51 group increased,with the largest proportion of in situ delete and the presence of ectopic delete;Compared with the tau-V337M sgRNA+Cas9 protein group,the type of InDel in the tau-V337M sgRNA+Cas9 protein+ssODN group increased,with the largest proportion of in situ delete,followed by in situ insert and ectopic delete;Compared with the tau-V337M sgRNA+Cas9 protein group,the type of InDel in the tau-V337M sgRNA+Cas9 protein+Rad51+ssODN group increased,with in situ insertion being the dominant,followed by in situ delete,and ectopic insertion being present.For V337M,the addition of Rad51 significantly increased the type of InDel,but the tau-V337M sgRNA+Cas9 protein+Rad51+ssODN group was the only one among the four groups that experienced precise point mutations(KIA>G).Part II:Establishment of a single base mutant mouse model of MeCP2-T158M and tau-V337M.Experimental results at the embryonic level show that the addition of Rad51 protein can significantly improve the efficiency of HDR.We used this method to establish a mouse model with single base mutations in MeCP2-T158M and tau-V337M.After bilateral fallopian tube transplantation of MeCP2-T158M two-cell embryos,four FO generation mice were obtained,one of which died on the day of birth.Sanger sequencing results showed that of the three surviving FO generation mice,two mice underwent gene knockout,one simultaneously underwent gene insertion and target site mutation,and one died on the day of birth was a gene knockin mouse,it is equivalent to changing the base site corresponding to threonine(ACG)at position 158 to a base encoding methionine(ATG).After unilateral fallopian tube transplantation of tau-V337M two-cell embryos,two F0 generation mice were obtained.After two weeks,the mouse tail genome was identified.Sanger sequencing results showed that in two F0 generation mice,one gene was knocked out and one gene was knocked in,equivalent to changing the base site corresponding to valine(GTG)at position 337 to the base encoding methionine(ATG).Part Ⅲ:Analysis of tau-V337M and MeCP2-T158M single base mutant mouse models.For tau-V337M single base mutant mice,first expand the population,and then conduct targeted testing on F0 and F1 tau-V337M single base mutant mice,in order to ensure that InDel caused by sgRNA targeting does not have an impact on animal phenotypes.The results showed that none of the four model mice missed the target at the three missed sites.Pathological analysis was performed on 6-month-old F0 generation model mice.After performing phosphorylated tau protein specific antibody AT8 and neuron specific antibody NeuN immunofluorescence staining on tissue sections of the mouse motor cortex,it was found that compared with wild mice of the same age,tauV337M point mutation mice had a large number of nerve fiber tangled filaments formed by tau protein hyperphosphorylation in the neuronal enrichment region.For T158M mice,behavioral evaluation was conducted on the 35th day of the acquisition of three F0 generation mutant mice.All three mutant mice exhibited gait disharmony,insufficient activity,tremor,and hind limb hugging.Further behavioral testing was conducted using a rat and mouse rotary fatigue tester and open field experiments.The results showed that the activity of T158M gene knockout mice was significantly reduced compared to wild type mice,Compared with wild-type mice,mice with T158M gene insertion and target site mutations also had a certain decrease in activity,but higher than T158M gene knockout mice.Three F0 mice died on 44d,48d,and 67d,respectively.Western blot(WB)was used to detect MeCP2 protein in T158M gene knockout mice and T158M point mutant mice.The results showed that compared with the control group,there was no MeCP2 protein expression in the brain of T158M gene knockout mice,and there was a small amount of MeCP2 protein expression in T158M point mutant mice.Immunofluorescence staining using protein specific antibody MeCP2 in T158M knockout mice showed no expression of MeCP2 protein in the brain of T158M knockout mice.Overall,our research demonstrates that chain exchange protein Rad51 can significantly enhance recombination efficiency and improve ssODN based HDR.At the same time,the study also found that the addition of Rad51 not only improves efficiency,but also increases the complexity of deletion and insertion mutation types caused by Cas9 cutting.Secondly,the two mutation models we obtained demonstrate that the combination of CRISPR/Cas9,ssODN,and Rad51 is efficient for the production of point mutation models located on autosomes or sex chromosomes.In addition,the distance between the PAM site and the target mutation site may affect the efficiency of Rad51 mediated HDR.The point mutation model mice at specific sites were obtained through the above methods,providing an accurate and reliable model for the research and gene therapy of related diseases. |
