CRISPR-Cas9D10A nickase-assisted base editing in the solvent producer Clostridium beijerinckii

Li, Qi; Seys, François M.; Minton, Nigel P.; Yang, Junjie; Jiang, Yu; Jiang, Weihong; Yang, Sheng

Abstract

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 D10A 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, spo0A, and araR. The efficiency of the system was such that they could sometimes be directly obtained following transformation, otherwise only requiring one single restreaking 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 knockout plasmids. This both reduces plasmid size and makes their construction simpler, for example, whereas the assembly of pNICKclos2.0 requires six primers for the assembly of a typical knockout 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.

Citation

Li, Q., Seys, F. M., Minton, N. P., Yang, J., Jiang, Y., Jiang, W., & Yang, S. (2019). CRISPR-Cas9D10A nickase-assisted base editing in the solvent producer Clostridium beijerinckii. Biotechnology and Bioengineering, 116(6), 1475-1483. https://doi.org/10.1002/bit.26949

• Journal Article Type: Article
• Acceptance Date: Feb 6, 2019
• Online Publication Date: Feb 10, 2019
• Publication Date: Feb 10, 2019
• Deposit Date: May 1, 2019
• Publicly Available Date: May 1, 2019
• Journal: Biotechnology and Bioengineering
• Print ISSN: 0006-3592
• Electronic ISSN: 1097-0290
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 116
• Issue: 6
• Pages: 1475-1483
• DOI: https://doi.org/10.1002/bit.26949
• Keywords: Biotechnology; Applied microbiology and biotechnology; Bioengineering
• Public URL: https://nottingham-repository.worktribe.com/output/2004151
• Publisher URL: https://onlinelibrary.wiley.com/doi/full/10.1002/bit.26949
• Additional Information: This is the peer reviewed version of the following article: Li, Q, Seys, FM, Minton, NP, et al. CRISPR–Cas9D10A nickase‐assisted base editing in the solvent producer Clostridium beijerinckii. Biotechnology and Bioengineering. 2019; 1– 9, which has been published in final form at https://doi.org/10.1002/bit.26949 This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions.
• Contract Date: May 1, 2019