Chemistry-Biology Interface Graduate Program, Johns Hopkins University, Baltimore, MD, United States of America.
Department of Chemical & Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, United States of America.
PLoS One. 2022 Jun 3;17(6):e0269270. doi: 10.1371/journal.pone.0269270. eCollection 2022.
The engineering of switchable or activatable dCas9 proteins would benefit from a single system for both positive and negative selection of dCas9 activity. Most systems that are used to interrogate dCas9 libraries use a fluorescent protein screen or an antibiotic selection for active dCas9 variants. To avoid some of the limitations of these systems, we have developed a single system capable of selecting for either active or inactive dCas9 variants. E. coli expressing active dCas9 variants are isolated in the positive selection system through growth in the presence of ampicillin. The negative selection can isolate cells lacking dCas9 activity through two separate mechanisms: growth in M9 minimal media or growth in media containing streptomycin. This system is capable of enriching for rare dCas9 variants up to 9,000-fold and possesses potential utility in directed evolution experiments to create switchable dCas9 proteins.
可切换或可激活的 dCas9 蛋白的工程设计将受益于一个单一的系统，用于正负选择 dCas9 的活性。大多数用于研究 dCas9 文库的系统都使用荧光蛋白筛选或抗生素选择活性 dCas9 变体。为了避免这些系统的一些限制，我们开发了一个能够同时选择活性或非活性 dCas9 变体的单一系统。在氨苄青霉素存在的情况下生长，表达活性 dCas9 变体的大肠杆菌在阳性选择系统中被分离出来。负选择可以通过两种独立的机制分离缺乏 dCas9 活性的细胞：在 M9 最小培养基中生长或在含有链霉素的培养基中生长。该系统能够将稀有 dCas9 变体富集 9000 倍以上，在定向进化实验中具有潜在的应用价值，可用于创建可切换的 dCas9 蛋白。
