CRISPR-Endogenous Cas Systems
CRISPR-Endogenous Cas Systems
Download the plasmid maps here
Attempts at introducing vectors containing the Cas9-encoding gene into Acetobacterium woodii have previously been unsuccessful, largely due to the presence of a restriction- modification system recognition site and the toxicity of the nuclease itself. We therefore developed an alternative approach which relies on the presence of an endogenous Type I-B CRISPR/Cas systems and delivery of the appropriate gRNA and appropriate homology arms to generate the knock-out or knock-in as part of the basic vector.
Vectors needed for the genome engineering of Acetobacterium woodii and Clostridium autoethanogenum based on their endogenous Type I-B CRISPR/Cas systems supplied as a kit of six vectors (pMTLMPD15; pMTLMPD21; pMTLMPD23; pMTLMPD24; pMTLMPD25; pMTLMPD28) which are available HERE.
(i) Acetobacterium woodii endogenous CRISPR kit
All A. woodii vectors (HERE) contain the catP gene for chloramphenicol or thiamphenicol selection and the pCD6 Gram-positive replicon. The four vectors supplied are: (a) pMTL-MPD15: empty cloning vector for construction of KO and KI plasmids, carrying the A. woodii leader sequence and a MCS for insertion of spacer and homology arms; (b) pMTL-MPD21: positive control for generating a pheA KO; (c) pMTL-MPD23: cloning vector for cargo delivery at the pheA locus, through its cloning into the MCS, and; (d) pMTL-MPD24: positive control for cargo delivery at the pheA locus through carriage of the FAST reporter gene flanked by the thiolase (thl) promoter (Pthl) and pyrE-hydA (orotate phosphoribosyltransferase and hydrogenase I) terminator from Clostridium acetobutylicum.
(ii) Clostridium autoethanogenum endogenous CRISPR kit
All C. autoethanogenum vectors (HERE) contain the catP gene for chloramphenicol or thiamphenicol selection and the pCB102 Gram-positive replicon. The two vectors supplied are: (a) pMTL-MPD25: empty cloning vector, for construction of KO and KI vectors containing the C. autoethanogenum leader sequence and a MCS for insertion of spacer and homology arms, and; (b) pMTL-MPD28: positive control for pyrE gene KO.
References
[1] Poulalier-Delavelle M, Baker JP, Millard J, Winzer K, Minton NP. Endogenous CRISPR/Cas systems for genome engineering in the acetogens Acetobacterium woodii and Clostridium autoethanogenum. Front Bioeng Biotechnol. 2023; 11:1213236.
https//doi.org/10.3389/fbioe.2023.1213236
[2] Seys FM, Humphreys CM, Tomi-Andrino C, Li Q, Millat T, Yang S, Minton NP. Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations. Front Bioeng Biotechnol. 10;11:1211197.
https//doi.org/10.3389/fbioe.2023.1211197