| Gender control is closely related to the economic benefits of livestock production.In industries restricted by gender,such as dairy farms and laying hen factories,the economic benefits of females are much higher than that of males.For livestock and poultry factories that produce meat,due to the different requirements and responses of males and females to environmental and nutritional conditions,it is necessary to achieve Male and female rearing in groups is conducive to scientific management,improving production performance and carcass quality.Therefore,it is of great significance to effectively control the sex of livestock and poultry in domestic and foreign aquaculture production.In mammals,the male sex chromosome type is XY,which makes it possible to achieve artificially controllable parthenogenesis by separating X,Y sperm in mammalian production.In chickens,due to the unique sex chromosome system,it is impossible to produce controllable sex offspring by segregating sperm.Only some basic sex identification techniques such as anus turning,distribution of blood vessels or the establishment of a male and female matching system can be used to identify chickens of different genders.For identification and group feeding,all identified roosters will even be eliminated in the laying hen factory,which greatly increases the cost of breeding.Although studies have shown that methods such as serum injection and hormone stimulation can achieve sex control of chickens,they are not suitable for large-scale promotion due to cumbersome operations and high costs.No methodologies have been found for parthension sex control in chickens that can be used in production practices.Some researchers knocked in the CRISPR/Cas9 knockout system in mice,and established a system of parthenogenesis by knocking out early embryonic development genes in mice.Inspired by this,gene editing technology can be combined with key genes that affect chicken sex to establish a sex control system on chickens.Studies have shown that the Dmrt1 gene located on the Z chromosome can promote the development of chicken gonads to testis.By changing the expression level of Dmrt1 gene,it was determined that Dmrt1 plays an important role in male sex determination.In the development of females,estrogen is an indispensable factor for the formation of ovaries,of which aromatase is the main determinant,and Cyp19a1 is the gene encoding aromatase.The previous research of our group showed that after interfering with Cyp19a1.the ovary of female chicken embryos was reduced.Testis-like tissue structures appeared,demonstrating that Cyp19a1 is critical in chicken female development.Based on this,our research group focused on the Dmrt1 and Cyp19a1 genes,combined with gene editing technology,and hoped to establish a parthenogenetic chicken sex control model by knocking in the Dmrt1 and Cyp19a1 knockout systems.Therefore,in this study,Dmrt1 and Cyp19a1 genes were used as target genes,a knockout system was established by CRISPR/Cas9 technology,and the function of the system was verified in vitro and in vivo.In order to prevent the subsequent knockout system from excessive knockout due to long-term expression,or some side effects caused by off-target,this study added Tet-on induction system to the constructed Dmrt1 and Cyp19a1 knockout systems,and constructed chicken Dmrt1 and Cyp19a1 inducible Knockout system,the inducible knockout system was verified to have effects on sex differentiation in vitro and in vivo.The specific research results are as follows:（1）Construction of Cyp19a1 knockout system.According to the CDS region of Cyp19a1 gene,three target sites were designed and linked into the VK001-08 vector backbone to construct Cyp19a1-sgRNA1,Cyp19a1-sgRNA2,Cyp19a1-sgRNA3 knockout vectors respectively.Cyp19al-sgRNA1,Cyp19al-sgRNA2,Cyp19al-sgRN A3 knockout vectors all had knockout activity by SSA activity assay.The results of T7E1 digestion showed that the knockout activity of Cyp19a1-sgRNA1 was 53.93%,the knockout activity of Cyp19a1sgRNA2 was 73.20%,and the knockout activity of Cyp19a1-sgRNA3 was 68.64%.The vector Cyp19a1-sgRNA2 with the highest knockout activity was screened and named Cyp19a1-KO was used as a Cyp19a1 knockout vector for follow-up studies.The results of TA cloning showed that Cyp19a1-KO could knock out the target,and the knockout efficiency was 86.6%（13/15）.In order to verify the effect of Cyp19a1-KO on sex differentiation,in this study,firstly,in vitro experiments,Cyp19a1-KO was transfected into female Chick Embryo Fibroblast（CEF）,the expression of female-related genes Foxl2 and Esr1 was significantly downregulated（P<0.05）,the expression of male sex-related gene Dmrt1 was significantly increased（P<0.05）,and Sox9 and Amh were significantly up-regulated（P<0.01）.After Cyp19a1-KO was injected into the body,5.5 d and 18.5 d female chicken embryo leg meat was taken for T7E1 digestion and TA cloning.The results showed that Cyp19a1-KO could be knocked out in chicken embryo with a knockout efficiency of 46%（11/24）.qRT-PCR results showed that the expression of Cyp19a1 on 5.5 d and 18.5 d was extremely significantly down-regulated（P<0.01）,the expression of female sex-related genes,Foxl2 and Esr1 was also extremely significantly down-regulated（P<0.01）,and the male sex-related gene Dmrt1,Sox9 and Amh were significantly up-regulated（P<0.01）.The grayscale analysis of the WB results showed that the protein expression of CYP19A1 at 5.5 d and 18.5 d was significantly down-regulated compared with the control group（P<0.01）,and the expression of the female sex-related gene FOXL2 was significantly decreased（P<0.05）.The protein expression of related gene SOX9 was significantly increased（P<0.05）.The ELISA results showed that the estradiol level after Cyp19a1-KO injection was decreased compared with the normal female gonad level.All the above results indicated that the Cyp19a1 knockout system was successfully constructed,and the Cyp19a1 gene could be knocked out at the cellular and individual levels,thereby affecting the female sex differentiation of chickens.（2）Construction of Dmrt1 knockout system.Through the bioinformatics website,the target sites sgRNA1,sgRNA2 and sgRNA3 were designed according to the CDS region of the chicken Dmrt1 gene,and the vectors Dmrt1-sgRNA1,Dmrt1-sgRNA2 and Dmrt1-sgRNA3 were successfully constructed.The results of SSA activity showed that Dmrt1-sgRNA1,Dmrt1-sgRNA2,and Dmrt1-sgRNA3 all had knockout activity.Dmrt1-sgRNA1 was screened as Dmrt1 knockout vector by T7E1 digestion results in Dmrt1-sgRNA1（knockout activity 74.07%）,Dmrt1-sgRNA2（knockout activity 67.23%）,Dmrt1-sgRNA3（knockout activity 56.50%）,It was named Dmrt1-KO,and the knockout efficiency of Dmrt1-KO vector was 80%（12/15）detected by TA cloning.In order to verify the effect of Dmrt1-KO on male sex differentiation,in vitro qRT-PCR results showed that transfection of Dmrt1-KO could inhibit the expression of Dmrt1 in male CEF（P<0.05）,and then inhibited the male sex-related genes Amh（P<0.05）and The expression of Sox9（P<0.01）also promoted the expression of female-related genes Cyp19a1（P<0.05）,Foxl2（P<0.05）and Esr1（P<0.05）.After injecting Dmrt1-KO vector into chickens,5.5 d and 18.5 d male chicken embryo muscle tissue was taken for T7E1 digestion and TA cloning detection.It was found that Dmrt1-KO could knock out Dmrt1 in vivo with a knockout efficiency of 42%.（10/24）,qRT-PCR results showed that injection of Dmrt1-KO could promote the expression of female sex-related genes Cyp19a1,Foxl2 and Esr1 mRNA（P<0.01）,and inhibit the male sex-related gene Sox9 and Amh mRNA expression（P<0.01）.The results of WB grayscale analysis also showed that after injection of Dmrt1-KO,the expression of SOX9 protein was significantly decreased at 5.5 d（P<0.05）,and was extremely significantly down-regulated at 18.5 d（P<0.01）,while the expression of CYP19A1 protein was extremely significantly increased（P<0.01）,the expression of FOXL2 protein was significantly up-regulated（P<0.05）.Testosterone levels in the gonads were detected by ELISA,and it was found that testosterone levels were suppressed after injection of Dmrt1-KO.All the above results indicated that the Dmrt1 knockout system was successfully constructed in this study,and it was verified that the system could successfully knock out the Dmrt1 gene and affect the male sex differentiation both in vitro and in vivo.（3）Construction of chicken Dmrt1 and Cyp19a1 inducible knockout system.Using the pEGFP-N1 vector as a template,clone the EGFP element and connect it into the TRE-tight vector to construct a TRE-EGFP recombinant plasmid,which was co-transfected with the pTet-on-advanced vector containing the rtTA element into DF-1 cells,and Dox was added to induce Fluorescent expression was found later,but no fluorescent expression was found when Dox was not added.It was demonstrated that the Tet-on system can induce gene expression by Dox.Using the VK001-08 vector as a template,clone the Puro element,connect it to the pTet-on-advanced and connect the rtTA element with T2A,and amplify the rtTA containing the promoter and the Puro element from the successfully constructed pTet-on-Puro vector.TRE-EGFP vector,a single plasmid vector TRE-EGFP-rtTA-Puro was successfully constructed,qRT-PCR results showed that the expression of Puro in DF-1 cells transfected with TRE-EGFP-rtTA-Puro was significantly higher than that in the control group（P<0.01）,at the same time,the cells transfected with TRE-EGFP-rtTA-Puro were subjected to Puro screening.The results showed that the untreated DF-1 cells died after Puro screening,while the cells transfected with TRE-EGFP-rtTA-Puro finally Only cells expressing green fluorescence survived,demonstrating that the Puro element can play a screening role.The Cas9 element in VK001-08 was replaced with the EGFP element in TRE-EGFP-rtTA-Puro,and the vector TRE-Cas9-rtTA-Puro was successfully constructed.qRT-PCR detection showed that the expression of Cas9 was the highest when Dox was induced for 12 h.WB results showed that the expression of Cas9 was the highest when Dox was induced by 20μg/mL.The sgRNAs in Dmrt1-KO and Cyp19a1-KO were linked into the sgRNA-sy004 vector to construct Cyp19a1 and Dmrt1 single sgRNA vectors Cyp19a1-sgRNA and Dmrt1sgRNA,which were co-transfected with the PX45 8 vector,detected by qRT-PCR,and digested with T7E1 enzyme And TA clone detection confirmed that Cyp19a1/Dmrt1-sgRNA can guide the knockout of Cyp19a1/Dmrt1.T7E1 digestion assay showed that under the induction of different concentrations,the inducible Cyp19a1/Dmrt1 knockout system composed of Cyp19a1 and Dmrt1 single sgRNA vectors and TRE-Cas9-rtTA-Puro had the highest knockout efficiency under the induction of 20 μg/mL Dox.The results of TA cloning showed that the Cyp19a1/Dmrt1 knockout system was 80%（12/15）in induced knockout efficiency under the induction of 20 μg/mL Dox,which was similar to the results of TA clone of the Cyp19a1/Dmrt1 knockout system.In order to verify the effect of the inducible Cyp19a1/Dmrt1 knockout system on the differentiation of male and female,the inducible Cyp19a1 knockout system composed of TRECas9-rtTA-Puro vector and Cyp19a1-sgRNA vector was transfected into CEF and added Dox to induce,qRT-PCR results showed that the expression of Cyp19a1（P<0.01）,and the femalerelated genes Foxl2 and Esr1（P<0.05）were inhibited when Dox was added,and the male sexrelated genes Dmrt1（P<0.05）,Sox9（P<0.05）and The expression of Amh（P<0.05）was upregulated,while the gene expression induced by no Dox addition was not significantly different from the blank control group.In contrast,the qRT-PCR results of transfectioninduced Dmrt1 knockout system were reversed.The dual vector and Dox contained in the inducible Cyp19a1/Dmrt1 knockout system were mixed,and injected into chicken embryos through blunt end at 0d.48h later,the results of qRT-PCR and WB detection showed that the addition of Dox-induced chicken embryos had Cas9 expression,but not.Addition of Doxinduced chick embryos did not express Cas9.The chicken embryo muscle tissue was taken for T7E1 digestion and TA cloning.The results showed that the Cyp19a1/Dmrt1 knockout system induced by the addition of Dox could be knocked out in vivo with an efficiency of 45%（9/20）.5.5 d and 18.5 d chick embryo gonads were used for qRT-PCR,WB and ELISA detection.The results showed that the results of the experimental group induced by adding Dox were the same as the results of Cyp19a1-KO and Dmrt1-KO injection,while the results of the experimental group not induced by Dox were the same.There was no significant difference in the control group.It can be proved that the inducible knockout system of chicken Dmrt1 and Cyp19a1 can be successfully constructed,and Dmrt1 and Cyp19a1 can be knocked out successfully in vitro and in vivo under Dox induction,thereby affecting the sex differentiation of chickens. |
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