A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences

Seys, Fran�ois M.; Rowe, Peter; Bolt, Edward L.; Humphreys, Christopher M.; Minton, Nigel P.

Abstract

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. Phenotypic complementation of gene knockouts is an essential step in establishing function. Here, we describe a simple strategy for ‘gold standard’ complementation in which the mutant allele is replaced in situ with a wild type (WT) allele in a procedure that exploits CRISPR/Cas9. The method relies on the prior incorporation of a unique 24 nucleotide (nt) ‘bookmark’ sequence into the mutant allele to act as a guide RNA target during its Cas9-mediated replacement with the WT allele. The bookmark comprises a 23 nt Cas9 target sequence plus an additional nt to ensure the deletion is in-frame. Here, bookmarks are tailored to Streptococcus pyogenes CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in Clostridium autoethanogenum. Complementation efficiencies reached 91%. As complemented strains are indistinguishable from their progenitors, concerns over contamination may be satisfied by the incorporation of ‘watermark’ sequences into the complementing genes.

Citation

Seys, F. M., Rowe, P., Bolt, E. L., Humphreys, C. M., & Minton, N. P. (2020). A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences. Genes, 11(4), Article 458. https://doi.org/10.3390/genes11040458

• Journal Article Type: Article
• Acceptance Date: Apr 23, 2020
• Online Publication Date: Apr 23, 2020
• Publication Date: Apr 23, 2020
• Deposit Date: Aug 17, 2020
• Publicly Available Date: Aug 28, 2020
• Journal: Genes
• Print ISSN: 2073-4425
• Electronic ISSN: 2073-4425
• Publisher: MDPI
• Peer Reviewed: Peer Reviewed
• Volume: 11
• Issue: 4
• Article Number: 458
• DOI: https://doi.org/10.3390/genes11040458
• Keywords: bookmark; CRISPR/Cas9; complementation; Clostridium; knock-out
• Public URL: https://nottingham-repository.worktribe.com/output/4336552
• Publisher URL: https://www.mdpi.com/2073-4425/11/4/458