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CRISPR-Cas9~(D10A) nickase-assisted base editing in solvent producer Clostridium beijerinckii

李琦  Fran?ois M.Seys  Nigel P.Minton  Junjie Yang  Yu Jiang  Weihong Jiang  杨晟  
【摘要】:Clostridium beijerinckii is a potentially important industrial microorganism as it can synthesize valuable chemicals and fuels from various carbon sources. The establishment of convenient to use, effective gene tools with which the organism can be rapidly modified is essential if its full potential is to be realized. Here, we developed a genomic editing tool(pCBEclos) for use in C. beijerinckii based on the fusion of cytidine deaminase(Apobec1), Cas9 D10 A nickase and uracil DNA glycosylase inhibitor(UGI). Apobec1 and UGI are guided to the target site where they introduce specific base-pair substitutions through the conversion of C·G to T·A. By appropriate choice of target sequence, these nucleotide changes are capable of creating missense mutation or null mutations in a gene. Through optimization of pCBEclos, the system derived, pCBEclos-opt, has been used to rapidly generate four different mutants in C. beijerinckii, in pyrE, xylR, spo0 A and araR. The efficiency of the system was such that they could sometimes be directly obtained following transformation, otherwise only requiring one single re-streaking step. Whilst CRISPR-Cas9 nickase systems, such as pNICKclos2.0, have previously been reported in C. beijerinckii, pCBEclos-opt does not rely on homologous recombination, a process that is intrinsically inefficient in clostridia such as C. beijerinckii. As a consequence, bulky editing templates do not need to be included in the knock-out plasmids. This both reduces plasmid size and makes their construction simpler, e.g., whereas the assembly of pNICKclos2.0 requires six primers for the assembly of a typical knock-out plasmid, pCBEclos-opt requires just two primers. The pCBEclos-opt plasmid established here represents a powerful new tool for genome editing in C. beijerinckii, which should be readily applicable to other clostridial species.

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