外源基因在转基因动物和细胞中稳定、高效的表达，对于基因功能的研究和生物反应器的建立至关重要。动物基因组中的友好基因座能使外源基因高效稳定的表达且无副作用，但目前猪基因组中可供外源基因安全、高效定点整合的基因座相对较少，限制了多转基因猪的研究和发展。本研究提出了一种将猪I型胶原α1链（collagen type I alpha 1 chain，COL1A1）基因座作为友好基因座表达外源基因的新策略。利用CRISPOR软件设计了一对靶向COL1A1基因终止密码子的sgRNA，并连接到CRISPR/Cas9表达载体pX330中；同时合成了一个不含启动子、左右同源臂各长为900 bp的2A-GFP供体载体，然后共转染猪肾上皮细胞（porcine kidney epithelial，PK15)，胎儿成纤维细胞（porcine embryonic fibroblast，PEF）以及回肠上皮细胞（porcine intestinal epithelial，IPI-2I）3种细胞。电转染48 h后观察3种细胞的荧光情况，72 h后通过流式细胞术分选绿色荧光蛋白阳性的细胞，并通过荧光定量、蛋白质印记、转录组测序和CCK8实验在不同水平上评估COL1A1基因作为猪基因组外源基因定点整合位点的安全性。结果表明，共转染sgRNA和供体载体之后，可以分别在猪3种不同细胞中观察到绿色荧光，说明同源重组介导的定点整合系统可以在猪基因组中很好的发挥作用。qPCR结果显示，GFP敲入对3种细胞COL1A1基因mRNA的表达并无显著影响（PK15、PEF和IPI-2I细胞中的P值分别为0.29、0.66和0.20）。同样，蛋白质印记结果显示，GFP阳性细胞与野生型细胞COL1A1蛋白的表达并无显著差异（PK15、PEF和IPI-2I细胞中的P值分别为0.64、0.48和0.80)。转录组测序结果显示，GFP阳性PEF细胞与野生型PEF细胞的转录组显著正相关（P<2.2e-16），表明GFP敲入没有改变内源性基因的整体表达。CCK8实验表明，GFP敲入对PK15细胞增殖并无显著影响（24 h，48 h，72 h，96 h，120 h的P值分别为0.31，0.96，0.24，0.17和0.38）。上述结果表明，COL1A1基因可作为猪基因组中外源基因定点整合的友好基因座，可广泛应用于家畜育种和生物医学模型的建立。本研究首次鉴定出了COL1A1基因可以作为猪基因组中的友好基因座。

Efficient and stable expression of foreign genes in cells and transgenic animals is important for gain-of-function studies and the establishment of bioreactors.  Safe harbor loci in the animal genome enable consistent overexpression of foreign genes, without side effects.  However, relatively few safe harbor loci are available in pigs, a fact which has impeded the development of multi-transgenic pig research.  We report a strategy for efficient transgene knock-in in the endogenous collagen type I alpha 1 chain (COL1A1) gene using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system.  After the knock-in of a 2A peptide-green fluorescence protein (2A-GFP) transgene in the last codon of COL1A1 in multiple porcine cells, including porcine kidney epithelial (PK15), porcine embryonic fibroblast (PEF) and porcine intestinal epithelial (IPI-2I) cells, quantitative PCR (qPCR), Western blotting, RNA-seq and CCK8 assay were performed to assess the safety of COL1A1 locus.  The qPCR results showed that the GFP knock-in had no effect (P=0.29, P=0.66 and P=0.20 for PK15, PEF and IPI-2I cells, respectively) on the mRNA expression of COL1A1 gene.  Similarly, no significant differences (P=0.64, P=0.48 and P=0.80 for PK15, PEF and IPI-2I cells, respectively) were found between the GFP knock-in and wild type cells by Western blotting.  RNA-seq results revealed that the transcriptome of GFP knock-in PEF cells had a significant positive correlation (P<2.2e–16) with that of the wild type cells, indicating that the GFP knock-in did not alter the global expression of endogenous genes.  Furthermore, the CCK8 assay showed that the GFP knock-in events had no adverse effects (P_24h=0.31, P_48h=0.96, P_72h=0.24, P_96h=0.17, and P_120h=0.38) on cell proliferation of PK15 cells.  These results indicate that the COL1A1 locus can be used as a safe harbor for foreign genes knock-in into the pig genome and can be broadly applied to farm animal breeding and biomedical model establishment