Abstract
BackgroundZymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. Recently, an endogenous CRISPR-based genome editing tool has been developed for this species. However, a simple and high-efficient genome editing method is still required.ResultsWe developed a novel gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway. This tool only requires a self-interference plasmid carrying the mini-CRISPR (Repeat–Spacer–Repeat) expression cassette, where the spacer matches the target DNA. Transformation of the self-interference plasmid leads to target DNA damage and subsequently triggers the endogenous MMEJ pathway to repair the damaged DNA, leaving deletions normally smaller than 500 bp. Importantly, the MMEJ repair efficiency was increased by introducing mutations at the second repeat of the mini-CRISPR cassette expressing the guide RNA. Several genes have been successfully deleted via this method, and the phenotype of a σ28 deletion mutant generated in this study was characterized. Moreover, large fragment deletions were obtained by transformation of the self-interference plasmids expressing two guide RNAs in tandem.ConclusionsHere, we report the establishment of an efficient gene deletion tool based on the endogenous subtype I–F CRISPR-Cas system and the MMEJ pathway in Zymomonas mobilis. We achieved single gene deletion and large-fragment knockout using this tool. In addition, we further promoted the editing efficiency by modifying the guide RNA expression cassette and selecting lower GC% target sites. Our study has provided an effective method for genetic manipulation in Z. mobilis.
Highlights
Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries
The Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is composed of CRISPR arrays and an endonuclease protein or several endonuclease proteins, whose DNA-targeting specificity and cutting activity can be programmed by a short guide RNA [12]
We developed a genetic tool based on the endogenous CRISPR-Cas system and the microhomology-mediated end joining (MMEJ) pathway to manipulate the genes in vivo by transformation of a shuttle vector only carrying the “Repeat-Spacer-Repeat” expression cassette, providing a simple and quick method to evaluate the gene functions in vivo
Summary
Zymomonas mobilis is a natural ethanologen with many desirable characteristics, making it an ideal platform for future biorefineries. To develop Z. mobilis as a model microbe for synthetic biology and biorefinery applications, a series of genome-editing approaches have been explored These approaches include classical chemical mutagenesis and adaptation, transposon mutagenesis, shuttle vectors and transformation approaches [6,7,8,9]. Clustered Regularly Interspaced Short Palindromic Repeats and its associated protein (CRISPR-Cas) systems were widely developed as genomic editing tools in bacteria [10, 11]. The endogenous I–F system showed higher efficiency than the heterogenous CRISPR-Cas or Cpf systems in Z. mobilis [19], providing a powerful toolkit for diverse genome engineering purposes, including gene mutation, large-fragment deletion, and simultaneous multiple gene editing, which greatly benefit further study in this species
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