Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 32611, United States.
University of Florida Genetics Institute, Gainesville, Florida 32610, United States.
ACS Synth Biol. 2023 Jul 21;12(7):1989-2003. doi: 10.1021/acssynbio.3c00065. Epub 2023 Jun 27.
Genome editing tools, through the disruption of an organism's native genetic material or the introduction of non-native DNA, facilitate functional investigations to link genotypes to phenotypes. Transposons have been instrumental genetic tools in microbiology, enabling genome-wide, randomized disruption of genes and insertions of new genetic elements. Due to this randomness, identifying and isolating particular transposon mutants (i.e., those with modifications at a genetic locus of interest) can be laborious, often requiring one to sift through hundreds or thousands of mutants. Programmable, site-specific targeting of transposons became possible with recently described CRISPR-associated transposase (CASTs) systems, allowing the streamlined recovery of desired mutants in a single step. Like other CRISPR-derived systems, CASTs can be programmed by guide-RNA that is transcribed from short DNA sequence(s). Here, we describe a CAST system and demonstrate its function in bacteria from three classes of Proteobacteria. A dual plasmid strategy is demonstrated: (i) CAST genes are expressed from a broad-host-range replicative plasmid and (ii) guide-RNA and transposon are encoded on a high-copy, suicidal pUC plasmid. Using our CAST system, single-gene disruptions were performed with on-target efficiencies approaching 100% in Beta- and Gammaproteobacteria ( and , respectively). We also report a peak efficiency of 45% in the Alphaproteobacterium . In , we performed simultaneous co-integration of transposons at two different target sites, demonstrating CAST's utility in multilocus strategies. The CAST system is also capable of high-efficiency large transposon insertion totaling over 11 kbp in all three bacteria tested. Lastly, the dual plasmid system allowed for iterative transposon mutagenesis in all three bacteria without loss of efficiency. Given these iterative capabilities and large payload capacity, this system will be helpful for genome engineering experiments across several fields of research.
基因组编辑工具通过破坏生物体的天然遗传物质或引入非天然 DNA，促进了将基因型与表型联系起来的功能研究。转座子是微生物学中重要的遗传工具，能够实现全基因组随机基因破坏和新遗传元件的插入。由于这种随机性，鉴定和分离特定的转座子突变体（即那些在感兴趣的遗传基因座上发生修饰的突变体）可能非常繁琐，通常需要筛选数百个甚至数千个突变体。最近描述的 CRISPR 相关转座酶 (CASTs) 系统使转座子的可编程、位点特异性靶向成为可能，允许在单个步骤中简化所需突变体的回收。与其他 CRISPR 衍生系统一样，CASTs 可以通过从短 DNA 序列转录的向导 RNA 来编程。在这里，我们描述了一个 CAST 系统，并证明了它在三类 Proteobacteria 细菌中的功能。展示了一种双质粒策略：（i）CAST 基因由广谱复制质粒表达，（ii）向导 RNA 和转座子编码在高拷贝、自杀性 pUC 质粒上。使用我们的 CAST 系统，在 Beta-和 Gammaproteobacteria（和 ，分别）中，单基因缺失的靶向效率接近 100%。我们还报告了 Alphaproteobacterium 中的峰值效率为 45%。在 中，我们在两个不同的靶位点同时进行了转座子的共整合，证明了 CAST 在多基因座策略中的实用性。CAST 系统还能够在所有三种测试的细菌中进行高效的大转座子插入，总插入长度超过 11 kbp。最后，双质粒系统允许在所有三种细菌中进行迭代转座子诱变，而不会降低效率。鉴于这些迭代能力和大容量有效载荷，该系统将有助于跨几个研究领域的基因组工程实验。
