Ayo Tolulope E, Xu Hao
Center for Molecular and Cellular Biosciences, School of Biological, Environmental, and Earth Sciences, University of Southern Mississippi, Hattiesburg, Mississippi 39406, United States of America.
J Genome Ed Regul. 2022;2. doi: 10.32371/jger/246146. Epub 2022 Dec 17.
CRSPR/Cas9-mediated base editing introduces point mutations in cellular DNA by exploiting target-specific single guide RNA (sgRNA) along with a genetically modified Cas9. Existing plasmid vectors for sgRNA expression in base editing contain either a fluorescent marker or an antibiotic resistance cassette but not both, preventing simultaneous monitoring and enrichment of transfected host cells. In this study, we introduced a fluorescent marker into pGL3-U6-sgRNA-PGK-puromycin, a popular sgRNA expression vector available at Addgene. Specifically, the cDNAs of mRFP and a T2A linker were inserted in between the hPGK promoter and the puromycin resistance gene (PuroR). After correct insertion was verified by DNA sequencing, this new plasmid, pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR, was utilized to generate a stop codon in the second exon of the Munc13-1 gene in RBL-2H3 cells. Both the mRFP fluorescent marker and the puromycin resistance marker functioned accordingly in the process. This new sgRNA vector therefore represents a useful addition to the CRISPR tool kit.
CRISPR/Cas9介导的碱基编辑通过利用靶向特异性单向导RNA（sgRNA）以及经过基因改造的Cas9在细胞DNA中引入点突变。现有的用于碱基编辑中sgRNA表达的质粒载体要么含有荧光标记，要么含有抗生素抗性盒，但不会同时具备两者，这就阻碍了对转染宿主细胞的同步监测和富集。在本研究中，我们将荧光标记引入了pGL3-U6-sgRNA-PGK-嘌呤霉素，这是一种可从Addgene获得的常用sgRNA表达载体。具体而言，mRFP的cDNA和一个T2A接头被插入到人磷酸甘油酸激酶（hPGK）启动子和嘌呤霉素抗性基因（PuroR）之间。经DNA测序验证插入正确后，这个新质粒pGL3-U6-sgRNA-PGK-mRFP-T2A-PuroR被用于在RBL-2H3细胞的Munc13-1基因的第二个外显子中产生一个终止密码子。在此过程中，mRFP荧光标记和嘌呤霉素抗性标记均发挥了相应作用。因此，这个新的sgRNA载体是CRISPR工具套件中一个有用的补充。
