Effects of homologous arm length on the efficiency of CRISPR/ Cas9 mediated hLF gene knock-in at goat β-lactoglobulin locus

【Objective】To explore the effect of homology arm length on the efficiency of CRISPR/Cas9 system-mediated human lactoferrin gene（hLF）targeting goat β-lactoglobulin gene（BLG）locus，and to provide a scientific basis for the preparation of BLG ^-/hLF ⁺ gene target cells by somatic cell nuclear transfer in the future. It also provided a reference for genetic breeding of BLG gene or other gene locus directed by precise molecular modification mediated by CRISPR/ Cas9 gene editing system.【Method】According to the first exon region of the goat BLG gene，the sgBLG/Cas9 vector was designed and constructed，and it was electrotransfected goat fetal fibroblasts. The site-mutagenic activity of the BLG gene locus was verified by PCR. Based on BLC14 mammary gland-specific expression vector，three hLF gene targeting vectors with homology arm length（6.0，3.5 and 1.2 kb）were constructed，and it was cotransfected into goat fetal fibroblasts with sgBLG/Cas9 vector respectively. After 500 µg/mL G418 screening，the gene targeting was detected by PCR.【Result】 The mutagenic activity efficiency of cleaving DNA double strands with BLG locus in goat fetal fibroblasts was 30%-35% by sgBLG/Cas9 vector. The hLF gene targeting vectors（BLC14-1，BLC14-2 and BLC14-3）were constructed to obtain three homology arm lengths，corresponding to 6.0，3.5 and 1.2 kb. Three hLF gene targeting vectors and sgBLG/Cas9 vector were cotransfected into goat fetal fibroblasts. After 5 times of electrotransfections and G418 screening，83，77 and 86 drug-resistant cells were obtained respectively. Finally，42，38 and 44 BLG ^-/hLF ⁺ gene targeting cell lines were obtained by PCR homologous recombination detection. The average targeting efficiencies of hLF gene at the BLG locus of goat fetal fibroblasts were 50.6%（42/83），49.4 %（38/77）and 51.2%（44/86）. There was no statistically significant difference in the targeting efficiency of hLF gene targeting vectors constructed with three different lengths of homology arms at the BLG gene locus in goats（P>0.05），indicating that the homology arm length had no significant effect on CRISPR/ Cas9-mediated hLF gene targeting at the BLG gene locus in goats.【Conclusion】A number of BLG ^-/hLF ⁺ gene targeting cell lines（BLG loci for site-directed target hLF genes）have been successfully obtained by CRISPR/Cas9 system-mediated hLF gene targeting at BLG gene locus in goat fetal fibroblasts. However，the length of the homology arm of the targeting vector has no significant effect on the targeting efficiency of the CRISPR/Cas9 system-mediated BLG site-directed integration of the hLF gene.