TY - JOUR
T1 - Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-Mediated Counter-Selection
AU - Corts, Anna D.
AU - Thomason, Lynn C.
AU - Gill, Ryan T.
AU - Gralnick, Jeffrey A.
N1 - Publisher Copyright:
© 2019 American Chemical Society.
PY - 2019/8/16
Y1 - 2019/8/16
N2 - Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≤5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent S. oneidensis mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in Shewanella genomic engineering.
AB - Dissimilatory metal-reducing bacteria, particularly those from the genus Shewanella, are of importance for bioremediation of metal contaminated sites and sustainable energy production. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Here we report the development of a highly efficient system based on single-stranded DNA oligonucleotide recombineering coupled with CRISPR/Cas9-mediated counter-selection. Our system uses two plasmids: a sgRNA targeting vector and an editing vector, the latter harboring both Cas9 and the phage recombinase W3 Beta. Following the experimental analysis of Cas9 activity, we demonstrate the ability of this system to efficiently and precisely engineer different Shewanella strains with an average efficiency of >90% among total transformed cells, compared to ≤5% by recombineering alone, and regardless of the gene modified. We also show that different genetic changes can be introduced: mismatches, deletions, and small insertions. Surprisingly, we found that use of CRISPR/Cas9 alone allows selection of recombinase-independent S. oneidensis mutations, albeit at lower efficiency and frequency. With synthesized single-stranded DNA as substrates for homologous recombination and Cas9 as a counter-selectable marker, this new system provides a rapid, scalable, versatile, and scarless tool that will accelerate progress in Shewanella genomic engineering.
KW - CRISPR/Cas9
KW - Shewanella
KW - genome editing
KW - recombineering
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U2 - 10.1021/acssynbio.9b00188
DO - 10.1021/acssynbio.9b00188
M3 - Article
C2 - 31277550
AN - SCOPUS:85070722760
SN - 2161-5063
VL - 8
SP - 1877
EP - 1889
JO - ACS Synthetic Biology
JF - ACS Synthetic Biology
IS - 8
ER -