目的:探讨运用CRISPR/Cas9基因编辑技术高效构建热休克因子1（heat shock factor 1，HSF1）基因敲除的小鼠模型，为小鼠原发骨肉瘤模型建立前期基础。方法:根据Hsf1基因结构的第9外显子选择相应的gRNA（guide RNA）并进行筛选，通过PCR扩增出sgRNA（small guide RNA）转录模版，得到4个上游引物。随后将sgRNA进行体外转录并通过TubeScreen平台筛选，筛选切割有效的sgRNA，从筛选结果中选取切割效率最高的sgRNA用于后续实验。运用PCR扩增出转录spCas9mRNA的模板，接着体外转录Cas9mRNA。将体外转录得到的sgRNA与Cas9mRNA一起注射入健康的C57BL/6小鼠受精卵内，剪取出生小鼠尾巴提取组织并进行PCR测序鉴定。选取F0代杂合子母鼠与野生型公鼠交配得到F1代子鼠，结合琼脂凝胶电泳和基因测序检测F1代鼠基因碱基的突变情况。选取F1代杂合子公鼠与F0代母鼠回交得到F2代子鼠，剪取鼠尾组织并测序，最终得到F2代纯合子敲基因小鼠。运用PCR观察sgRNA切割效率与鼠尾组织的测序，通过观察凝胶电泳结果来判断小鼠是否为杂合，同时利用snapgene软件进行基因序列比对判断碱基片段缺失。结果:经PCR扩增得到上游引物sgRNA-1primer-F、sgRNA-2primer-F、sgRNA-3primer-F、sgRNA-4primer-F与下游引物sgRNAprimer-R。经过sgRNA的体外转录和筛选，sgRNA-1, sgRNA-2和sgRNA-4切割效率高，被选取用于后续实验。将T7promoter添加到Cas9mRNA的5’端，运用PCR和体外转录试剂盒得到Cas9mRNA。把混合的Cas9-sgRNA溶液注入到小鼠受精卵中并进行培养。把培养至二细胞的受精卵注射到假孕母鼠的壶腹部，并成功获得F0代小鼠。通过琼脂凝胶电泳结果判断共得到F0代杂合子小鼠8只。将F0代3号杂合子母鼠与健康的野生型公鼠繁育后，结合PCR与测序比对，共得到F1代杂合子基因敲除小鼠3只。将三只F1代杂合子公鼠与F0代3号母鼠回交，得到F2代小鼠。通过对F2代小鼠电泳和测序比对的结果观察，证实7只小鼠缺失Hsf1碱基序列，电泳结果为突变型条带且不存在野生型条带，鉴定为纯合子；得到的F2代纯合子小鼠能够稳定繁育，结果证明本实验成功建立Hsf1基因敲除小鼠模型。结论:成功运用CRISPR/Cas9技术构建Hsf1基因敲除小鼠模型，稳定性强，可重复性高，为进一步研究Hsf1基因表达产物及原发骨肉瘤小鼠模型的建立奠定了基础。“,”Objective:To explore the efficient construction of HSF1 gene knockout mouse model using CRISPR/Cas9 gene editing technology, and to establish the early basis for the mouse model of primary osteosarcoma.Methods:According to exon 9 of HSF1 gene structure, the corresponding GRNA (guideRNA) was selected and screened. Then the transcription template of sgRNA (small guide RNA) was amplified by PCR, and four up stream primers were obtained. Subsequently, sgRNA was transcribed in vitro and screened by Tube Screen platform to screen the sgRNA with effective cutting, and the sgRNA with the highest cutting efficiency was selected from the screening results for subsequent experiments. The transcription template of SPCas9mRNA was amplified by PCR, and then Cas9mRNA was transcribed in vitro. The sgRNA transcribed in vitro and Cas9mRNA were injected into the fertilized eggs of healthy C57BL/6 mice, and the tissue was extracted from the tail of the born mice and identified by PCR sequencing. Heterozygous female mice of F0 generation were selected to mate with wild-type male mice too btain F1 generation off spring. The mutation of gene bases of F1 generation mice was detected by AGAR gel electrophoresis and gene sequencing. The heterozygous male mice of the F1 generation and female mice of the F0 generation were back crossed to obtain the F2 generation daughter mice. The tail tissues were cut and sequenced to obtain the F2 generation homozygous knockout mice. PCR was used to observe the cutting efficiency of sgRNA and the sequencing of rat tail tissue, and SNAPGene software was used for gene sequence alignment to determine the deletion of base fragments.Results:The up stream primers sgRNA-1 Primer-f, sgRNA-2 Primer-f, sgRNA-3 Primer-f, sgRNA-4 Primer-f and down stream primers sgRNA-4 Primer -r were obtained by PCR amplification. After in vitro tran scription and screening of sgrRNA, sgrRNA-1, sgrRNA-2 and sgrRNA-4 had high cleavage efficiency and were selected for subsequent experiments. T7 promoter was added to the 5 'end of Cas9 mRNA, and Cas9 mRNA was obtained by PCR and in vitro transcription kit. Mixed Cas9-sgRNA solution was injected into the fertilized eggs of mice and cultured. The cultured two-cell fertilized eggs were injected into the ampulla of the pseudo pregnant female mice, and the F0 generation mice were obtained successfully. A total of 8 heterozygous mice of F0 generation were obtained by Agar gel electrophoresis. Three heterozygous knockout mice of F1 generation were obtained by breeding the female heterozygous mice of F0 generation with healthy wild-type male mice and PCR and sequencing. Three heterozygous male mice of F1 generation were back crossed with female mice of F0 generation 3 to obtain F2 generation mice. Through the observation of electrophoresis and sequencing results of F2 generation mice, it was confirmed that 7 mice were missing HSF1 base sequence, and the electrophoresis results showed mutant bands and no wild-type bands, which were identified as homozygous. The F2 generation homozygous mice were able to breed stably. As eries of results proved that the HSF1 gene knockout mouse model was successfully established in this experiment.Conclusion:CRISPR/Cas9 technology was successfully used to construct HSF1 gene knockout mouse model, with strong stability and high reproducibility, which laida foundation for further study of HSF1 gene expression products and establishment of mouse model of primary osteosarcoma.