Genetic engineering of Acidithiobacillus ferridurans with CRISPR-Cas9/dCas9 systems

Genus Acidithiobacillus includes a group of Gram-negative Fe/S-oxidizing acidophilic chemolithotrophic bacteria that are extensively studied and used for biomining processes. Synthetic biology approaches are key means to study and improve their biomining performance. However, efficient genetic manipulations in Acidithiobacillus are still major bottlenecks. In this study, we report a simple and efficient pAFi system (CRISPR-dCas9) and a scarless pAF system (CRISPR-Cas9) for genetic manipulations in A. ferridurans JAGS. The pAFi system harboring both dCas9 and sgRNA was constructed based on pBBR1MCS-2 to knockdown HdrA and TusA genes, separately, of which the transcription levels were significantly downregulated by 48% and 93%, separately. The pAF system carrying pCas9-sgRNA-homology arms was constructed based on pJRD215 to delete HdrB3 gene and overexpress Rus gene. Our results demonstrated that the pAF system is a fast and efficient genome editing method with an average rate of 15-20% per transconjugant in one recombination event, compared to 10-3 and then 10-2 in two recombination events by traditional markerless engineering strategy. Moreover, with these two systems, we successfully regulated iron and sulfur metabolisms in A. ferridurans JAGS: the deletion of HdrB3 reduced 48% of sulfate production, and substitution overexpression of Rus promoter showed 8.82-fold of mRNA level and enhanced iron oxidation rate. With these high-efficient genetic tools for A. ferridurans, we will be able to study gene functions and create useful recombinants for biomining applications. Moreover, these systems could be extended to other Acidithiobacillus strains and promote the development of synthetic biology-assisted biomining. HighlightsO_LITwo shuttle vectors were constructed for Acidithiobacillus ferridurans C_LIO_LIAll-in-one pAFi (CRISPR-dCas9) and pAF (CRISPR-Cas9) systems were built up for gene knockdown and genome editing, separately C_LIO_LIThe transcription levels of HdrA and TusA were reduced 48% and 93% using pAFi system and thus suppressed sulfur oxidation C_LIO_LIHdrB3 deletion and Rus overexpression were achieved using pAF system and showed significant effects on sulfur and iron oxidation respectively C_LIO_LIOur pAF system facilitated genome editing in Acidithiobacillus ferridurans with high efficiency (15-20%) in less than 4 weeks C_LI