Co-expression of two distinct guide RNAs (gRNAs) has been used to facilitate the application of CRISPR/Cas9 system in fields such as large genomic deletion. The paired gRNAs are often placed adjacently in the same direction and expressed individually by two identical promoters, constituting direct repeats (DRs) which are susceptible to self-homologous recombination. As a result, the paired-gRNA plasmids cannot remain stable, which greatly prevents extensible applications of CRISPR/Cas9 system. To address this limitation, different DRs-involved paired-gRNA plasmids were designed and the events of recombination were characterized. Deletion between DRs occurred with high frequencies during plasmid construction and subsequent plasmid propagation. This recombination event was RecA-independent, which agreed with the replication slippage model. To increase plasmid stability, a reversed paired-gRNA plasmids (RPGPs) cloning strategy was developed by converting DRs to the more stable invert repeats (IRs), which completely eliminated DRs-induced recombination. Using RPGPs, rapid deletion of chromosome fragments up to 100 kb with an efficiency of 83.33% was achieved in Escherichia coli. The RPGPs cloning strategy serves as a general solution to avoid plasmid RecA-independent recombination. It can be adapted to applications that rely on paired gRNAs or repeated genetic parts.

中文翻译：

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反向配对gRNA质粒克隆策略，可在大肠杆菌中进行有效的基因组编辑。

共表达两种不同的指导RNA（gRNA）已被用于促进CRISPR / Cas9系统在大基因组缺失等领域的应用。成对的gRNA通常在同一方向上相邻放置，并由两个相同的启动子分别表达，构成易于自身同源重组的直接重复（DR）。结果，成对的gRNA质粒不能保持稳定，这极大地阻止了CRISPR / Cas9系统的可扩展应用。为了解决这个限制，设计了不同的DR参与的成对的gRNA质粒，并对重组事件进行了表征。在质粒构建和随后的质粒繁殖期间，DR之间的删除频率很高。此重组事件是独立于RecA的，与复制滑移模型一致。为了增加质粒的稳定性，通过将DR转化为更稳定的反向重复序列（IR），开发了反向配对gRNA质粒（RPGPs）克隆策略，这完全消除了DRs诱导的重组。使用RPGPs，在大肠杆菌中可以快速删除高达100 kb的染色体片段，效率为83.33％。RPGPs克隆策略可作为避免质粒RecA独立重组的通用解决方案。它可以适应依赖于成对的gRNA或重复的遗传部分的应用。在大肠杆菌中达到33％。RPGPs克隆策略是避免质粒RecA独立重组的通用解决方案。它可以适应依赖于成对的gRNA或重复的遗传部分的应用。在大肠杆菌中达到了33％。RPGPs克隆策略可作为避免质粒RecA独立重组的通用解决方案。它可以适应依赖于成对的gRNA或重复的遗传部分的应用。