| Research background and ideasGene knockout technology referred to the use of certain technical method to delete or inactivate specific genes.Currently,the widespread used gene knockout methods included zinc finger nuclease(ZFN),transcription activator-like effector nuclease(TALEN),CRISPR/Cas9 etc.The main principle is to cut specific DNA sequence utilizing endonuclease,and it will cause use gene frameshift mutation,substitution or deletion based on the characteristics of homologous recombination repair or non-homologous end repair when DNA double-strand is breaking,ultimately leading to the loss of gene function.With the development of genetic engineering technology,the novel,high efficient and accurate CRISPR/Cas technology are increasingly appreciated by scholars.Compared with other two technologies,the CRISPR/Cas9 can edit multiple genes at the same time without major species restriction.Sex Hormone-Binding Globulin(SHBG)is a 90-100 KDa homodimeric glycoprotein which is encoded by a single gene on the short arm of chromosome 17 and has two identical peptide chains.It is primarily produced by the liver and then secreted into the bloodstream and it functions as a circulating plasma protein to bind estrogen and testosterone,regulate their bioactivity,metabolic clearance and plasma level,but also control their entry into tissues and target cells.Furthermore,the binding of SHBG to the receptor in cell membrane plays an important role in signaling transduction,sex hormones especially 5a-dihydrotestosterone(DHT)can increase intracellular cAMP and then activate PKA by interacting with the SHBG-R-SHBG complex in membrane,its downstream effects seemingly included inhibiting the proliferation of MCF-7 cells mediated by estrogen,promoting the growth of prostate cancer cells ALVA-41,prohibiting the growth promoting effect of prostate cancer cells LNCaP caused by DHT etc.Recent studies had proved that plasma SHBG levels fluctuated in several diseases including obesity,metabolic syndrome,polycystic ovary syndrome(PCOS),and Cushing syndrome,indicating it can be used as a biomarker to guide the diagnosis of clinical diseases.Current basic researches are usually limited to using conventional Si SHBG mRNA method to knockdown SHBQ and lacking of SHBG gene knock out animal models puts a brake on further researches.In this study,CRISPR/Cas technology was used to successfully construct a SHBG gene knockout rat model and confirmed the reliability of SHBG knockout at the DNA and protein levels.Subsequently,in order to understand the growth,development and reproductive status of SHBG knockout rats,further provide a stable animal model for illustrating the role of SHBG in sex hormone levels regulating,reproductive behavior managing,tumorigenesis and developing,we detected the body weight,organ weight,blood routine indicators,blood biochemistry indicators,sex hormone indicators and reproductive ability of rats of different genotypes.High-throughput sequencing experiments screened out 165 differentially expressed genes between homozygous and wild-type rat groups,and further weighted co-expressed gene network analysis(WGCNA)identified 4 genes which were significantly related to sex hormone levels and SHBG expression(LOC108348081,STXBP5L,CLEC2DL1,SNORD26),suggested that these genes may coordinate with SHBG to regulate the level of sex hormones,providing new insights and directions for the future study about the specific mechanism of SHBG involving in sex hormone levels modulating.Materials and Method1 Establishment of SHBG gene knockout rat model1.1 All experimental rats were F344 rats,which were purchased from Beijing Viewsolid Biotechnology Co.Ltd.The structure of rat SHBG gene was analyzed by NCBI Genebank.Based on the genome structure and protein function conserved regions of SHBG,combined with the basic principles of gene knockout,the online target prediction website http://crispor.tefor.net/is used to predict the appropriate knockout site.1.2 We obtained four gRNA targets using the prediction tool,then transcribed the selected gRNA in vitro utilizing the spCas9-gRNA target efficiency detection kit.Following this,we prepared the dsDNA that needs to be digested.We recognized the genomic DNA of SHBG as a template,the DNA fragments with gRNA targets are amplified by PCR,and the results of enzyme digestion are detected by agarose gel electrophoresis.Each enzyme digestion reaction requires a standard target gRNA digestion reaction,and the most suitable gRNA target is selected by comparing the activity.1.3 Firstly,we put a ligated male mouse and a mature female mouse in the same cage.If a yellow and white plug is detected in the female mouse's vaginal,the female mouse is in a pseudopregnant state.Then we selected female mice of appropriate age for superovulation treatment,collected oocytes and then performed in vitro fertilization to obtain fertilized eggs.After mixing gRNA and Cas9 mRNA,we used a microinjection device to inject the mixture into the cytoplasm of the fertilized egg.Finally,we chose high-quality fertilized eggs to inject into the opening of the fallopian tube umbrella of the pseudopregnant female mouse.Genotype identification and sequencing analysis were performed on the born rats,and the F0generation SHBG+/-rats were screened.1.4 We predicted off-target sites using the CRISPR target design online website(http://crispor.tefor.net/crispor.py),and we selected the top four potential off-target sites for verification.We designed primers based on the gene sequence at the off-target site to perform PCR,and then performed high-throughput sequencing on the PCR product to verify the off-target situation.2 Identification and breeding of SHBG gene knockout ratsFirstly,SHBG+/-rats of FO generation and wild-type F344 rats were mated in cages,and F1 generation rats were obtained.After toe cutting and numbering,DNA was extracted for genotype identification,and SHBG+/-rats were screened out.After they were raised to sexual maturity,the reproductive system were expanded by self-crossing SHBG+/-rats.After the rat is born,the toe is cut and sequenced,and three F2 genotype rats of SHBG-/-,SHBG+/-and SHBG WT are obtained.3 The expression of SHBG protein in liver and blood of SHBG gene knockout ratsNine F2 generation rats of 3 lines and nine F2 generation rats of 10 lines were selected for verification,among which three were heterozygous,three were homozygotes and another three were wild-type rats.The liver of the rats were collected for protein extraction.At the same time,blood was collected to extract total protein.Then we detected the expression of SHBG utilizing Western blot.4 Phenotype observation of SHBG gene knockout rats4.1 The body weight and various organs weights of SHBG knockout ratsEighty F2 generation genotype rats were selected,including 20 homozygous,30 heterozygous and 30 wild-type rats.The body weight of each rat was measured from 24 weeks of age,and recorded every two weeks for a total of 10 times until it was 44 weeks of age.In addition,24 F2 rats of different genotypes were selected at 28 weeks of age.After the rats were fasted for 12-16 hours,the brain,heart,lung,liver,kidney,spleen,uterus,ovaries,testes and other organs were taken,and the surface water was absorbed by filter paper.After weighing,put it into the cryotube and store it in liquid nitrogen.4.2 Determination of various physiological and biochemical indexes of SHBG knockout ratsThe eighty rats whose body weights were monitored as described above were tested by taking blood from the orbit at the age of 24 weeks,36 weeks and 48 weeks.Routine blood tests require anti coagulated blood samples.An automatic blood cell analyzer was used to detect 23 blood routine indicators of the three genotypes of rats;the blood samples were centrifuged and the supernatant was collected to detect 12 blood biochemical indicators of the rats using an automatic biochemical analyzer.ELISA method was used to determine the serum estrogen and testosterone levels.5 SHBG knockout rats breeding experimentWe selected F2 generation male rats and 2-3 female rats to breed in the same cage.Seeing a female rat with plug means a successful breeding.The cumulative number of female rats for each male rat should not exceed three.After seeing the plug,the female rats need to be reared,marked and recorded separately.After delivery,the date of delivery,the number of offspring rats and the ratio of male to female should be recorded.6 Transcriptome sequencing analysis of SHBG gene knockout rats6.1 Transcriptome sequencingAt the transcriptome level,six rats of each of the three genotypes were selected,and liver tissues were taken.Homozygous rats were named "Homozygous group",heterozygous rats were named "Heterozygote group",and wild-type rats were named"Wild type group",after extracting total RNA,we prepared library preparations and sequenced on an Illumina Novaseq platform to complete differentially expressed gene analysis.Adjust P value<0.05 and |log2FoldChange|>1 were selected as the standard to screen for significant difference genes,and then functional annotation,GO enrichment analysis and KEGG pathway enrichment analysis were performed on the differentially expressed genes.Gene set enrichment(GSEA)analysis was performed using sequencing data of wild-type and homozygous rats.6.2 WGCNA analysisFPKM data obtained from 18 rat transcriptome sequencing were used to perform weighted gene co-expression network analysis(WGCNA),and R package WGCNA were applied for co-expression network construction,topological feature calculation,data simulation and visualization,and module determination.Combined with the sex hormone test results of 48-week-old rats,traits are associated with gene modules.Then we identified gene modules related to sex hormone levels.We extracted genes in the module of interest for GO enrichment analysis and KEGG pathway enrichment analysis.6.3 Correlation analysisKey genes were obtained by extracting the genes in the above-mentioned key modules and intersecting the differentially expressed genes between the wild-type group and the homozygous group.In order to further screen the genes related to SHBQ we used transcriptome data to extract the expression of 14 genes and SHBQ then we used Pearson correlation coefficient to concuct correlation analysis.7 Statistical analysisSPSS 21.0 was used for statistical analysis of the experimental results.The data were displayed as meanąstandard deviation,and differences between groups were analyzed by t test or chi-square test.R v3.6.1 was used to statistically analyze and compare the litter size of the three groups of rats,and the Wilcoxon test was used to analyze and compare the differences between two groups.The package 'Corrplot R'is used for correlation analysis,and the test method is Pearson's correlation coefficient method.The R package 'GO plot' is used for enrichment analysis of differentially expressed genes.GraphPad 8.0 software was applied to make graphs.Results1 Establishment of SHBG gene knockout rat modelAccording to the prediction results of the website,four gRNA targets with the highest predicted knockout efficiency were obtained.The activity test results showed that SHBG-gRNA2 has the highest activity.DNA sequencing of newborn rats of the F0 generation showed that two strains of SHBG knockout rats were obtained.In the knockout site of 3 strains of rats,an A is inserted before CTGTTCTGACCATT.In the knockout site of 10 strains of rats,2 bases(CA)are deleted before CTGTTCTG ACCATT.Due to gene insertion or deletion,the stop codon TGA appears in advance,which shortens the protein sequence,and because the deletion position is located before the Laminin G domain of the SHBG protein functional domain,the SHBG gene can be successfully knocked out.2 Breeding and identification of SHBG gene knockout rat modelAfter a year of breeding and expansion,A total of 29 litters of rats were bred from F0 and F1 generations.There were 87 rats in F1 generation and 184 rats in F2 generation.The homozygous rate of F2 generation was about 28.8%.Each offspring rat was identified by genetic sequencing.The sequencing results indicated that the missing sequences of the SHBG+/-and SHBG-/-genotype rats of the two strains of F2 generation were consistent with those of the F0 generation.In addition,western blot showed that SHBG protein is normally expressed in liver and blood of wild-type rats,and its expression level is reduced in heterozygotes,while its expression is absent in knockout rats.3 Phenotype observation of SHBG gene knockout rats3.1 Body weight monitoring of SHBG knockout ratsThe results of body weight monitoring showed that the weight of homozygous female mice aged 24-44 weeks was significantly higher than that of wild-type female mice,while the weight of homozygous male mice aged 24-44 weeks was significantly lower than that of heterozygous male mice and wild-type male mice.3.2 SHBG gene knockout rat blood biochemical indicatorsThe results of blood routine monitoring in 24,36,and 48-week-old rats showed that basically all routine blood indexes were not significantly different between rats of different genotypes in the three time periods,and only a few indexes such as neutrophil count eosinophil count,lymphocyte count had significant differences.We speculated that various environmental factors have affected the physiological state of rats,which caused this difference.The differences in blood biochemical indexes are affected by age rather than genotype.Moreover,the liver and kidney functions of homozygous rats at 24 weeks were abnormal compared with wild-type rats(decreased total protein and increased creatinine),while other indicators at various time points were not significantly different between rats of different genotypes.In general,knocking out SHBG has no significant effect on the physiological state of rats,and homozygous rats are normal.3.3 Monitoring of sex hormone levels in SHBG gene knockout ratsThe results of continuous monitoring of sex hormone indicators of the three genotypes of rats(at 24,36 and 48 weeks of age)showed that the serum concentration of estrogen in wild-type rats first increased and then decreased,reaching the highest level at 36 weeks.The difference is statistically significant.In homozygous and heterozygous,its concentration level changes in the same trend,but the difference is not statistically significant.The concentration of testosterone in wild-type and heterozygous rats also increased first and then decreased,reaching the highest level at 36 weeks,and the difference was statistically significant.But in homozygous rats,testosterone levels have been decreasing,and the difference is not statistically significant.In addition,the difference of the two hormones among rats of different genotypes was not statistically significant.4 SHBG knockout rat breeding experimentThe results of breeding experiments showed that SHBG gene deletion did not cause infertility in female or male mice,and the health status of newborn mice was not significantly different from that of wild type.The statistical analysis of the self-crossing results of the three genotypes showed that the number of progeny rats obtained from self-crossing homozygous rats was significantly less than the number of progeny rats obtained from self-crossing wild-type rats.5 Transcriptome sequencing experiment of SHBG knockout ratsThe transcriptome sequencing quality test results met the standards,the reference genome was selected correctly,and the data was valid for further analysis.The results of differential gene analysis showed that the expression of 86 genes increased significantly in the wild-type group,and the expression of 79 genes decreased significantly in the wild-type group.The enrichment analysis of differentially expressed genes by GO and KEGG showed that about biological process,the differentially expressed mRNA were enriched in the regulation of TGF-?receptor signaling pathway and the cell response to cAMP.About the cell component,differentially expressed mRNA were enriched in items such as protein complexes involved in cell adhesion.About the molecular function,differentially expressed mRNA were enriched in items such as P53 binding and nuclear receptor activity.KEGG enrichment analysis showed differentially expressed mRNA were enriched in calcium reabsorption regulated by endocrine and other factors and cAMP signaling pathway.These results indicated that the knockout of SHBG gene may change mRNA expression and affect the activity of the above pathways.The results of GSEA analysis showed that the three functional sets including galactose metabolism in the wild-type group were enriched with P<0.05.Seven functional sets such as glutathione metabolism in the homozygous group were enriched with P<0.05.6 WGCNA analysisWGCNA analysis successfully constructed a co-expressed gene network and divided it into 43 modules,each of which represents an effective module.Correlation analysis with sex hormone levels showed that steelblue and blue modules are strongly correlated with estrogen and testosterone.GO and KEGG analysis results showed that the main function of the steelblue module is cell response to thyroid-stimulating hormone,JAK-STAT signal cascade reaction,etc.The main functions of the blue module were the glucose metabolism process,steroid metabolism process and other functions.KEGG analysis results showed that the signaling pathways related to this module mainly included estrogen signaling pathway and PI3K-Akt signaling pathway.We extracted the genes of the two key modules and intersected them with the different genes of the wild-type group and the homozygous group obtained above,14 key genes were obtained.Correlation analysis results indicated that SHBG was positively correlated with LOC108348081,and negatively correlated with STXBP5L,CLEC2DL1 and SNORD26.Conclusion1.Two lines of SHBG knockout rats were successfully constructed.Both DNA and protein levels proved that SHBG has been accurately knocked out and no off-target phenomenon occurred.2.The absence of SHBG would increase the body weight of female rats and decrease the weight of male rats.SHBG konck out did not affect the general physiological state of rats as well as the growth and development of vital organs,but only change the weight of female reproductive organs.The litter size per litter of homozygous rats was significantly lower than that of wild-type rats.3.The lack of SHBG does not cause abnormal serum sex hormone levels,but it participates in the regulation process of male sex hormone levels.There are differences in the regulation of estrogen or androgen by SHBG in rats of different genders.4.LOC108348081,STXBP5L,CLEC2DL1,and SNORD26 are not only related to SHBG at the mRNA level,but also correlated to the regulation of rat sex hormone levels. 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