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



Peter Rowe

Associate Professor

Professor of Applied Molecular Microbiology


© 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.


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.

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
Keywords bookmark; CRISPR/Cas9; complementation; Clostridium; knock-out
Public URL
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