Generation And Evaluation Of FGF5 Gene-edited Cashmere Goat With The Base Editing System

Based on the single-base gene editing system derived from CRISPR technology,with its unique base substitution function,theoretically,the conversion of cytosine C to thymine T is not introduced without the introduction of double-strand breaks in DNA and without the need for a homologous template.Compared with traditional CRISPR/Cas9 technology,which uses homologous recombination to complete single-base replacement,the operation of the single-base editing system has the advantages of simpler,wider application,higher base replacement efficiency,and fewer by-products.At the same time,many changes in livestock production traits and disease are caused by single nucleotide mutations.Therefore,it is of great significance to use single base editing system to construct large animal models to improve animal production traits.The FGF5 gene of Shanbei white cashmere goat is related to the growth and development of hair follicles.FGF5 protein inhibits the growth and development of the Shanbei white cashmere goat.After knocking out the FGF5 gene,the growth rate of Shanbei white cashmere goat can be accelerated and the amount of cashmere production can be increased.Therefore,in this experiment,Shanbei white cashmere goat was used as the test object,and a single base editor(Base Editor 3,BE3)was used to introduce a stop codon in the first exon region of the FGF5 gene in advance,and then the FGF5 gene was knocked out in goat functional expression in individuals.The test process and specific results are as follows:(1)The efficiency of base editor was verified in goat fetal fibroblasts.Four sg RNAs were designed on the first exon of the FGF5 gene,and a corresponding mammalian cell expression plasmid was constructed.The sg RNA and BE3 plasmids were co-transfected into goat fetal fibroblasts,and positive cells producing single base mutations were obtained.The editing efficiency of the four sg RNAs in fibroblasts was detected,and the editing efficiency was 10%-34%.It is shown that the designed sg RNAs can effectively target the FGF5 gene in fetal fibroblasts.(2)Preparation of FGF5 gene editing goat.The four sg RNA expression vectors were constructed in vitro,and the four sg RNA and BE3 vectors were transcribed in vitro to obtain the corresponding sg RNA m RNA and BE3 m RNA.Pregnant ewes were obtained through simultaneous estrus and natural mating,and embryos were collected.A mixture of BE3 m RNA and sg RNA m RNA was injected into 47 embryos at the one-cell stage.Embryos were injected with a mixture of BE3 m RNA and sg RNA m RNA.After in vitro culture,22 well-developed embryos were transplanted into 7 recipient ewes,3 were conceived,and 5 goats were finally generated(#3,#16,#18,#19,and # 25).(3)Detection of gene editing goat editing efficiency and off-target efficiency.It was found that the FGF5 gene of all offspring lambs was edited,and many sites had the expected efficient point mutations.Therefore,the editing efficiency is 100%.Off-target prediction and deep sequencing analysis were performed on the two gene-edited goats #16 and #25.Of 19 predicted off-target sites,one off-target site was found.Genetic analysis showed that the off-target site was caused by the BE3 system,rather than inherited from its parents.(4)Phenotypic analysis of gene-edited goats.Goat skins from edited and wild-type goats at the same age were collected and stained with hematoxylin and eosin.It was observed that the number of hair follicles of gene-edited goats was greater than that of wild-type goats.Comparing the length of cashmere fiber of gene-edited goats with wild-type goats at the age of 1 month and 2 months,it was found that the length of cashmere fiber of gene-edited goats was significantly longer than that of wild-type goats.Transcriptome sequencing showed that there was almost no difference in the m RNA expression of the FGF5 gene in the skins of gene-edited goats and wild-type goats.The expression of FGF5 protein in hip skin of gene-edited goats and wild-type goats at the same age was analyzed by immunofluorescence and protein immunoblot hybridization.The expression of FGF5 protein in gene-edited goats was lower than that in wild-type goats.(5)Detection of p53 cancer-related genes in goat skins.The expression of p53 protein in skin was detected using immunofluorescence and Western blotting.No difference was found between gene-edited goats and wild-type goats.This indicates that the BE3 modified gene-edited goats did not change the cancer risk compared with wild-type goats.In summary,this study successfully used the single-base editor BE3 to introduce a stop codon in the genome in advance to prepare the FGF5 gene knockout Shanbei white cashmere goat.This experiment proved the feasibility of using base editors on large mammals.This editing strategy can simply and quickly introduce point mutations and knock out target genes.