The fast-growing bacterium Vibrio natriegens has recently gained increasing attention as a novel chassis organism for a wide range of projects. To fully harness the potential of this fascinating bacterium, convenient and highly efficient genome editing methods are indispensable to create novel strains, tailored for specific applications. V. natriegens is able to take up free DNA and incorporate it into its genome by homologous recombination. This process, called natural transformation, was tamed for genome editing. It displays a high efficiency and is able to mediate uptake of multiple DNA fragments, thereby allowing multiple simultaneous edits. Here, we describe NT-CRISPR, a combination of natural transformation with CRISPR/Cas9 counterselection. In two temporally distinct steps, we first performed a genome edit by natural transformation and second, induced CRISPR/Cas9, targeting the wild type sequence, leading to death of non-edited cells. Through highly efficient cell killing with efficiencies of up to 99.999 %, integration of antibiotic resistance markers became dispensable and thus enabled scarless and markerless edits with single-base precision. We used NT-CRISPR for deletions, integrations and single-base modifications with editing efficiencies of up to 100 % and further demonstrated its applicability for the simultaneous deletion of multiple chromosomal regions. Lastly, we demonstrated that the near PAM-less Cas9 variant SpG Cas9 is compatible with NT-CRISPR and thereby massively broadens the target spectrum.

中文翻译：

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NT-CRISPR：结合自然转化和 CRISPR/Cas9 反选择，在 Vibrio natriegens 中进行无标记和无疤痕基因组编辑

快速生长的细菌Vibrio natriegens最近作为一种用于各种项目的新型底盘生物越来越受到关注。为了充分利用这种迷人细菌的潜力，方便且高效的基因组编辑方法对于创建针对特定应用量身定制的新菌株是必不可少的。V. natriegens能够吸收游离 DNA 并通过同源重组将其整合到其基因组中。这个过程被称为自然转化，用于基因组编辑。它显示出高效率并且能够介导多个 DNA 片段的摄取，从而允许多个同时编辑。在这里，我们描述了 NT-CRISPR，它是自然转化与 CRISPR/Cas9 反选择的结合。在两个时间上不同的步骤中，我们首先通过自然转化进行基因组编辑，然后诱导 CRISPR/Cas9，靶向野生型序列，导致未编辑的细胞死亡。通过高达 99.999% 的高效细胞杀伤，抗生素抗性标记的整合变得可有可无，从而实现了单碱基精度的无痕和无标记编辑。我们使用 NT-CRISPR 进行删除，整合和单碱基修饰的编辑效率高达 100%，并进一步证明了其同时删除多个染色体区域的适用性。最后，我们证明了接近无 PAM 的 Cas9 变体 SpG Cas9 与 NT-CRISPR 兼容，从而大大拓宽了目标谱。