TY - JOUR
T1 - Multiplex genome engineering in Clostridium beijerinckii NCIMB 8052 using CRISPR-Cas12a
AU - Patinios, Constantinos
AU - de Vries, Stijn T.
AU - Diallo, Mamou
AU - Lanza, Lucrezia
AU - Verbrugge, Pepijn L.J.V.Q.
AU - López-Contreras, Ana M.
AU - van der Oost, John
AU - Weusthuis, Ruud A.
AU - Kengen, Servé W.M.
PY - 2023/6/22
Y1 - 2023/6/22
N2 - Clostridium species are re-emerging as biotechnological workhorses for industrial acetone–butanol–ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.
AB - Clostridium species are re-emerging as biotechnological workhorses for industrial acetone–butanol–ethanol production. This re-emergence is largely due to advances in fermentation technologies but also due to advances in genome engineering and re-programming of the native metabolism. Several genome engineering techniques have been developed including the development of numerous CRISPR-Cas tools. Here, we expanded the CRISPR-Cas toolbox and developed a CRISPR-Cas12a genome engineering tool in Clostridium beijerinckii NCIMB 8052. By controlling the expression of FnCas12a with the xylose-inducible promoter, we achieved efficient (25–100%) single-gene knockout of five C. beijerinckii NCIMB 8052 genes (spo0A, upp, Cbei_1291, Cbei_3238, Cbei_3832). Moreover, we achieved multiplex genome engineering by simultaneously knocking out the spo0A and upp genes in a single step with an efficiency of 18%. Finally, we showed that the spacer sequence and position in the CRISPR array can affect the editing efficiency outcome.
U2 - 10.1038/s41598-023-37220-y
DO - 10.1038/s41598-023-37220-y
M3 - Article
C2 - 37349508
AN - SCOPUS:85162793938
SN - 2045-2322
VL - 13
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 10153
ER -