Readily curable plasmids facilitate the construction of plasmid-free bacterial strains after the plasmid encoded genes are no longer needed. The most popular of these plasmids features a temperature-sensitive (Ts) pSC101 origin of replication which can readily revert during usage and cannot be used to construct Ts mutations in essential genes. Plasmid pAM34 which contains an IPTG-dependent origin of replication largely overcomes this issue but is limited by carrying the most commonly utilized antibiotic selection and replication origin. This study describes the construction of an expanded series of plasmid vectors having replication origins of p15a, RSF1030 or RSF1031 that like pAM34 have IPTG-dependent replication. Surprisingly, these plasmids can be cured in fewer generations than pAM34. Derivatives of pAM34 with alternative antibiotic selection markers were also constructed. The utility of these vectors is demonstrated in the construction of a CRISPR-Cas9 system consisting of an IPTG-dependent Cas9 plasmid and a curable guide RNA plasmid having a streptomycin counterselection marker. This system was successfully demonstrated by construction of point mutations, deletions and insertions in the E. coli genome with a very high efficiency and in a shorter timescale than extant methods. The plasmids themselves were readily cured either together or singly from the resultant strains with minimal effort.

中文翻译：

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具有严格可抑制复制起点的大肠杆菌载体可以简化 Crispr/Cas9 基因编辑。


在不再需要质粒编码基因后，易于治愈的质粒有助于构建无质粒的细菌菌株。其中最受欢迎的质粒具有温度敏感 (Ts) pSC101 复制起点，在使用过程中很容易恢复，并且不能用于在必需基因中构建 Ts 突变。含有 IPTG 依赖性复制起点的质粒 pAM34 在很大程度上克服了这个问题，但由于携带最常用的抗生素选择和复制起点而受到限制。本研究描述了具有 p15a、RSF1030 或 RSF1031 复制起点的扩展系列质粒载体的构建，这些复制起点与 pAM34 一样具有 IPTG 依赖性复制。令人惊讶的是，这些质粒可以在比 pAM34 更少的代数内治愈。还构建了具有替代抗生素选择标记的 pAM34 衍生物。这些载体的实用性在 CRISPR-Cas9 系统的构建中得到了证明，该系统由 IPTG 依赖性 Cas9 质粒和具有链霉素反选择标记的可治愈指导 RNA 质粒组成。该系统通过在大肠杆菌基因组中构建点突变、缺失和插入而得到成功证明，与现有方法相比，效率非常高且时间更短。质粒本身可以很容易地从所得菌株中一起或单独地以最小的努力治愈。