| 娄 琪,张 菊,王 洁,张石生,张竞方.靶向AML突变的CRISPR/Cas9基因敲除文库构建[J].现代生物医学进展英文版,2021,(22):4201-4206. |
| 靶向AML突变的CRISPR/Cas9基因敲除文库构建 |
| Construction of a sgRNA Library Targeting AML Mutations |
| Received:March 31, 2021 Revised:April 27, 2021 |
| DOI：10.13241/j.cnki.pmb.2021.22.001 |
| 中文关键词: CRISPR/Cas9 sgRNA基因敲除文库 高通量测序 慢病毒包装 AML |
| 英文关键词: CRISPR/Cas9 sgRNA knockout library High-throughput sequencing Lentiviral packaging AML |
| 基金项目:国家自然科学青年基金项目（31701280） |
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| 中文摘要: |
| 摘要 目的：构建靶向约200个AML基因突变的sgRNA基因敲除文库，为进一步探索诱发AML的信号通路网络奠定基础。方法：TCGA对200名AML病人进行了全基因组或全外显子组测序，鉴定出约2000个AML相关基因突变，从中选出了约200个突变两次或以上的基因作为靶向基因；接着，从Brie文库中挑选出相应基因的sgRNA序列，每个基因对应4条sgRNA；利用Gibson组装酶连接到慢病毒载体内，得到sgRNA文库；之后，采用pSSA荧光素酶基因报告系统鉴定文库sgRNA的切割活性；对文库进行高通量测序鉴定；用慢病毒包装文库，并测定病毒滴度。结果：1、构建了一个靶向约200个AML突变的sgRNA基因敲除文库；2、pSSA荧光素酶基因报告系统鉴定文库sgRNA具有切割活性；3、鉴定的7个单克隆质粒序列完全正确；4、高通量测序鉴定文库丰度和均一性符合要求；5、用慢病毒包装成病毒文库，测定病毒文库滴度为4.4×107符合后续实验要求。结论：成功构建了靶向约200个基因突变的sgRNA敲除文库，可用于大规模地筛选诱发AML的基因突变，为探索AML发生、发展的分子机制以及药物靶点奠定基础。 |
| 英文摘要: |
| ABSTRACT Objective: In this research, we attempt to generate a sgRNA knockout library targeting approximately 200 AML genetic alterations identified in human patients. This library will be a useful tool to decipher the functional signaling pathways that synergistically induce AML. Methods: The TCGA database contains the genome sequences of 200 AML patients, from which around 2000 genetic alterations were identified. We selected approximately 200 genetic alterations that occurred at least twice in this database for further study. The sgRNAs were selected from the Brie library, 4 for each gene. A lentiviral library was constructed as a pool using Gibson assembly. High-throughput sequencing was performed to evaluate the quality of the library. Then, the library was packaged into a pool of lentivirus particles, and viral titer was measured. Results: 1. A sgRNA knockout library targeting about 200 AML genetic alterations was generated. 2. The cleavage activity of representative sgRNAs in the library was confirmed using pSSA reporter assay. 3. Plasmids from 7 randomly picked bacterial monoclones were prepared and verified by sequencing. 4. High-throughput sequencing demonstrated uniform distribution of sgRNA species in the library. 5. Lentiviral library was generated and viral titer (4.4×107) was high enough for further study. Conclusion: We generated a sgRNA knockout library targeting about 200 genetic mutations identified in AML patients, which can be used for pooled screening of AML driver mutations. This may help us to study molecular mechanisms of AML and to develop targeted therapies. |
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