US 11,746,346 B2
Genetic tool for the transformation of Clostridium bacteria
Francois Wasels, Metz (FR); Nicolas Lopes Ferreira, Croisilles (FR); Florent Collas, Aachen (DE); and Ana Lopez Contreras, Utrecht (NL)
Assigned to STICHTING WAGENINGEN RESEARCH, Pb Wageningen (NL); and IFP ENERGIES NOUVELLES, Rueil Malmaison (FR)
Appl. No. 15/768,018
Filed by IFP ENERGIES NOUVELLES, Rueil Malmaison (FR); and STICHTING WAGENINGEN RESEARCH, Wageningen (NL)
PCT Filed Oct. 14, 2016, PCT No. PCT/FR2016/052663
§ 371(c)(1), (2) Date Apr. 13, 2018,
PCT Pub. No. WO2017/064439, PCT Pub. Date Apr. 20, 2017.
Claims priority of application No. 1559846 (FR), filed on Oct. 16, 2015.
Prior Publication US 2018/0305680 A1, Oct. 25, 2018
Int. Cl. C12N 15/10 (2006.01); C12N 9/22 (2006.01); C12N 15/74 (2006.01); C12N 15/90 (2006.01); C12P 7/06 (2006.01); C12P 7/28 (2006.01); C12P 7/04 (2006.01); C12N 15/63 (2006.01); C12N 1/20 (2006.01); C12R 1/145 (2006.01)
CPC C12N 15/102 (2013.01) [C12N 1/205 (2021.05); C12N 9/22 (2013.01); C12N 15/63 (2013.01); C12N 15/74 (2013.01); C12N 15/902 (2013.01); C12P 7/04 (2013.01); C12P 7/065 (2013.01); C12P 7/28 (2013.01); C12R 2001/145 (2021.05); Y02E 50/10 (2013.01)] 11 Claims
 
1. A genetic tool allowing the transformation by homologous recombination of a solventogenic bacterium of the genus Clostridium, wherein said genetic tool comprises:
a first vector comprising a first nucleic acid sequence encoding at least Cas9, wherein the Cas9 coding sequence is placed under the control of a promoter, and
a second vector comprising a second nucleic acid sequence containing a repair template allowing, by a homologous recombination mechanism, the replacement of a portion of a Cas9- targeted bacterial DNA by a sequence of interest,
in that i) at least one of said first or second nucleic acid sequences further encodes one or more guide RNAs (gRNAs), or ii) the genetic tool further comprises one or more guide RNAs, each guide RNA comprising a Cas9-enzyme-binding RNA structure and a sequence complementary to the portion of the Cas9-targeted bacterial DNA, said complementary sequence comprising at least 10 nucleotides,
wherein Cas9 causes double-stranded cleavage of the bacterial DNA and the bacterial DNA is the DNA of a solvent-forming bacterium of the genus Clostridium.