Optimization Of CRISPR/Cas9 System For Goat Genome Editing

The recently emerged CRISPR/Cas9 technology has been widely used in animal gene editing.Compared with the previous gene editing technologies(e.g.ZFN and TELAN),the CRISPR/Cas9 system is simple to design,more efficient and specific.CRISPR/Cas9 system can be used in large-scale gene-modified animals generation process.However,several problems still existed in monotocous animals,such as off-target effects and low target efficiency.In order to improve the gene-modified efficiency and eliminate the off-target effect in goats and other large monotocous animals,we optimized the CRISPR/Cas9 systems which aimed to the FGF5 gene in goat.In this study,we optimized the sgRNA design process,CRISPR/Cas9 system microinjection content,and targeting strategy.We obtained a sgRNA,which targeting on the FGF5 gene of goat,with high targeting efficiency and no off-target effects.We examined the CRISPR/Cas9 system by optimizing micro-injection content and determined the optimum mixture.We found that co-injection of three sgRNA is able to improve the targeting efficiency and homozygous editing efficiency.The main process and results are as follows:(1)Optimization the sgRNA design process.We compared 18 sgRNA design software and found the most suitable computional tool for sgRNA design of goat is sgRNAcas9.We designed sgRNAs targeting on the first exon of FGF5 gene by sgRNAcas9,22 sgRNAs were obtained,and 8 sgRNAs with the lowest off-target risk and 2 sgRNAs we used in previous research was chosen for cell transfection.Cas9 mRNA and 10 sgRNAs were transfected individually to goat fibroblasts.We found that the sg8 had the highest efficiency and specificity.(2)Optimization of CRISPR/Cas9 micro-injection content.We set 13 different CRISPR/Cas9 micro-injection content groups,the Cas9 mRNA concentration gradient of test group is 10,20,50,100 ng/?L,the ratio of Cas9 mRNA and sgRNA is 20:1,4:1,2:1,and the control group only injected with RNase-free water.Approximately 25 embyros were injected for each group.A total of 362 one-cell stage zygotes were injected.Finally,we found that the highest targeting efficiency reached 66.7% when the concentration of Cas9 m RNA is 50 ng/?L and the ratio of Cas9 mRNA and sgRNA concentration is 2:1.(3)Optimization of the targeting strategy.We designed three sgRNAs to target the first exon of goat FGF5.Micro-injection of three sgRNA and Cas9 mRNA to one-cell stage embryos of goat,62 lambs were obtained.,and 32 lambs was detected with genome editing.We further showed that 28 of them were homozygous edited goats.These results indicated that co-injection of three sgRNAs with Cas9 mRNA can imporve the targeting efficiency and homozygous ratios in goats.In summary,we constructed the sgRNA optimizing system of goats,this can be used in goat and other large animals.We found the optimum CRISPR/Cas9 micro-injection content,laying the foundation for goat and other large animal genomic editing process.We proved that co-inject of multiple sgRNAs can improved the genomic editing efficiency and homozygous positive ratio of goats,this strategy is simple,robust and efficient,and can be widely applied to goat and other large animals for genomic modification studies.