US 11,946,067 B2
Optimized genetic tool for modifying Clostridium bacteria
Nicolas Lopes Ferreira, Croisilles (FR); François Wasels, Metz (FR); and Gwladys Chartier, Chatou (FR)
Assigned to IFP ENERGIES NOUVELLES, Rueil Malmaison (FR)
Filed by IFP ENERGIES NOUVELLES, Rueil Malmaison (FR)
Filed on May 24, 2019, as Appl. No. 16/421,572.
Claims priority of application No. 18 54835 (FR), filed on Jun. 4, 2018.
Prior Publication US 2019/0367947 A1, Dec. 5, 2019
Int. Cl. C12N 15/90 (2006.01); C12N 1/20 (2006.01); C12N 15/10 (2006.01); C12N 15/74 (2006.01); C12R 1/145 (2006.01)
CPC C12N 15/902 (2013.01) [C12N 1/20 (2013.01); C12N 1/205 (2021.05); C12N 15/102 (2013.01); C12N 15/74 (2013.01); C12N 2310/20 (2017.05); C12N 2830/00 (2013.01); C12R 2001/145 (2021.05)] 12 Claims
 
1. A genetic tool for improving efficiency of transformation over a gene editing tool that does not include anti-CRISPR proteins, and genetic modification by homologous recombination, of a bacterium of the genus Clostridium, characterized:
i) in that the genetic tool comprises:
a first nucleic acid encoding at least Cas9, wherein the nucleic acid encoding Cas9 is placed under the control of an inducible promoter, and
at least a second nucleic acid allowing the expression of a repair template comprising a sequence of interest allowing, by a homologous recombination mechanism, replacement of a portion of bacterial DNA targeted by Cas9 by the sequence of interest,
ii) in that at least one of said nucleic acids further encodes one or more guide RNAs (gRNAs) or in that 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 targeted portion of the bacterial DNA, and
iii) in that the first nucleic acid further comprises a sequence encoding an anti-CRISPR protein placed under the control of an inducible promoter, said genetic tool improving efficiency of transformation over a gene editing tool that does not include anti-CRISPR proteins.