Knocking Out Goat BLG Gene And Intergration Of Hlf Site-specifically Using CRISPR/Cas9 Technology

Goat milk with various nutrients is an ideal alternative of human milk.However,goat milk not only contains ?-lactoglobulin(BLG),a major milk allergen,but also lacks in lactoferrin which is abundant in human milk.Human lactoferrin is a multifuntional protein with various biological function,such as iron absorption in intestinal tract,antibacterial activity,antiviral activity,anticarcinogenic activity and antioxidant activity.Thus,it is an ideal food addition or medicine.Previous reported methods,for example somatic cell nucleus transfer(SCNT),have numerous disadvantages,including complicated technique,high expense,a long-time period,a low survival rate and disorder of development among off-springs.CRISPR/Cas9,as the most popular genome editing technology,can specifically modify genome of animals or plants,which is an ideal tool to generate gene modified animal.Nowadays,researchers have already generated gene KO or KI rabbits,pigs,sheep,goats and cows by using CRISPR/Cas9 technology.However,there is no report about knocking out goat BLG gene and intergration of hLF at BLG locus.Thus,we selected goats as the research object.Firstly,CRISPR/Cas9 mediated goat BLG gene targeting system was constructed.Secondly,knocking-in hLF gene at BLG locus efficiently using CRISPR/Cas9.Thirdly,exploration of availability of generation of BLG knock-out and hLF knock-in goats using microinjection of CRISPR/Cas9 system.Finally,the changes of milk protein gene expression levels in the mammary gland,milk quality and BLG protein expression level in the milk of the BLG knock-out goat has been detected.The main results were as follows:1.Development of CRISPR/Cas9 mediated goat BLG gene targeting systemIn order to investigate whether CRISPR/Cas9 system can be applied to edit goat BLG locus and tranfection efficiencies of different transfection methods.In present study,three sgRNAs targeting BLG locus were designed and inserted into Cas9-sgRNA expressing vector,including pCas9-sgl,pCas9-sg2 and pCas9-sg3,meanwhile,a BLG targeting vector pBHA-hLF-NIE was constructed.Then three Cas9-sgRNA expressing vector were tranfected into GEFs respectively.Cells were collected and DNA were extracted 72 h after transfection.The target region was PCR amplified and subjected into T7EN1 aasay for determine the editing efficiency.Also,the PCR product was ligated into T-vector and the mutations were detected using Sanger sequencing method.The?1500 bp 5'&3'homologous arms were PCR amplified from the goat genome and ligated into T-vector,naming pBHA-1.pBHA-hLF-NIE targeting vector was constructed,using pBHA-1 as a backbone vector,by inserting a NEO-IRES-EGFP fragment.The vector pBHA-hLF-NIE was transfected into GEFs via lipofection or electroporation respectively.72 h after transfection,cells were collected and tranfection efficiencies were analyzed through flow cytometric counting EGFP positive cell rate.Sequencing result suggested that the sgRNA were successfully inserted into pX330 and the targeting vector pBHA-hLF-NIE was constructed correctly.The flow cytometric analysis results showed that the transfection efficiency of electroporation is significantly higher than lipofection's(15.97%± 1.56 vs 2.38%± 0.10,P<0.05).T7EN1 assay results suggested that the editing efficiency of lipofection was beyond detection and efficiency of electroporation was 6.00%-10.0%.Sanger sequencing results showed that no mutation was found in lipofection group and 8%-12.5%of clones sequenced were with mutations.In all,these results suggested,(1)CRISPR/Cas9 system can edit goat BLG locus properly.(2)pBHA-hLF-NIE vector can express EGFP protein in GEFs,which could be applied in the following knock-in experiment.(3)Electroporation has a higher efficiency than lipofection and could be used for obtaining hLF knock-in cell strains in the following experiments.2.Knocking-in hLF at goat BLG locus using CRISPR/Cas9 and improving the KI efficiency with RS-1In order to investigate whether Rad51 stimulatory compound's(RS-1)can enhance the efficiency of homologous recombination,knock out goat ?-lactoglobulin(BLG)and knock in human lactoferrin(hLF)at BLG locus,we co-transfected goat ear fibroblasts(GEFs)with pCas9-sgl and pBHA-hLF-NIE vector.Then,transfected cells were treated with 0,5,10 and 20 ?M RS-1 for 72 h and analysed by flow cytometry after four passages.In addition,800 ?g/mL G418 were used to screen G418-resistent cells from GEFs treated with RS-1.Next,GFP positive single clones were picked and screened for hLF site-specific knock-in single clones by PCR and sequencing.Our results showed that the editing efficiency of sgl was 25%-31%.The adherent cells decreased sharply 72 h after treated in RS-1,especially in 20?M RS-1.The hLF knock-in efficiency is positively related to concentration of RS-1 in drug-free knock-in experiment,which were at most enhanced three-fold(?14%)when treated with 20 ?M RS-1.We got the highest hLF knock-in clone rate(32.61%)when 10 ?M RS-1 was used before G418 selection,however,the hLF knock-in positive rate dropped(22.22%)and number of senescent clones increased when the concentration of RS-1 was increased to 20 ?M.These results suggest we successfully obtain hLF knock-in and BLG knock-out GEFs via CRISPR/Cas9 system and moderate concentration of RS-1 can accelerate the knock-in efficiency significantly.3.Generation of BLG KO and hLF KI goats by CRISPR/Cas9In order to generation of BLG KO milk goat,Cas9 mRNA and sgRNAs were co-injected into one-cell stage embryos and the embryos were transfer to synchronized recipients.The newborn kids' DNA were extracted and genotypes were indentified by PCR amplification.What's more,the predicted off-target sites were PCR amplified and analyzed by T7EN1 assay.The efficiency of single sgRNA,lower concentration dual-sgRNA and higher concentration dual-sgRNA targeting were 6.66%,25.00%and 28.6%,respectively.And the hLF knock-in efficiency was 0%in both embryos and newborn kids.In addition,no editing activities were detected and the organs and tissues from dead BLG-modified goats were edited identically,some of which were chimeric.Our results suggested that micro-injection of Cas9 mRNA and sgRNAs into single-cell embryo is an efficient way to generate BLG KO goats and increasement of the concentration of Cas9 mRNA and sgRNA can obviously accelerate the targeting efficiency,however,could possibly induced chimerism.This study provided a base for generation of gene-edited livestocks via one-step method and further investigation of phenotype change of BLG protein knock-out goats.4.Analysis of the expression levels of milk protein genes in the mammary gland and BLG protein in milk of the BLG KO goatIn order to analyze changes of BLG protein expression in milk and milk gene expression level in mammary gland,the BLG KO goat and wildtype goats were hormonally induced for lactation and the milk samples and mammary gland tissues were collected.Then DNA and mRNA of the mammary were extracted and cDNA of mammary glands was reverse-transcribed.Secondly,sgRNA target region of gDNA and cDNA were PCR amplified and ligated into T vector for sequencing.Thirdly,the milk protein gene expression levels were analyzed by qRT-PCR.Fourthly,the milk qulity was analyzed by automatic milk quality analyzer.Last,the whey proteins were separated by SDS-PAGE electrophoresis and stained by Coomassie blue and BLG expression in milk was detected by Western Blot.Our qRT-PCR result demonstrated that the expression level of BLG dropped significantly(P<0.01)in mammary gland,meanwhile,other milk protein gene(CSN1S1,CSN1S2,CSN2,CSN3 and LALBA)expression levels decreased significantly(P<0.05)as well.Coomassie blue staining result showed that the BLG protein band had been abolished,which was confirmed by Western Blot.Our research had analyzed the milk quality,milk protein gene expression lvels in the mammary gland and BLG expression in milk of BLG knock-out goat,which proved that biallelic knock-out of BLG gene could successfully abolish BLG protein in goat milk.These results provided some valuable data for generation BLG-free goat milk in the future.