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Open AccessConcept Paper

A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences

Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK
School of Life Sciences, Queen’s Medical Centre, Nottingham NG7 2UH, UK
NIHR Nottingham Biomedical Research Centre, Nottingham University Hospitals NHS Trust, Nottingham NG7 2RD, UK
Author to whom correspondence should be addressed.
Present address: Deep Branch Biotechnology, Nottingham NG8 1AA, UK.
Genes 2020, 11(4), 458;
Received: 3 March 2020 / Revised: 9 April 2020 / Accepted: 20 April 2020 / Published: 23 April 2020
(This article belongs to the Special Issue CRISPR-Cas: Interactions with Genome and Physiological Maintenance)
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. View Full-Text
Keywords: bookmark; CRISPR/Cas9; complementation; Clostridium; knock-out bookmark; CRISPR/Cas9; complementation; Clostridium; knock-out
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MDPI and ACS Style

Seys, F.M.; Rowe, P.; Bolt, E.L.; Humphreys, C.M.; Minton, N.P. A Gold Standard, CRISPR/Cas9-Based Complementation Strategy Reliant on 24 Nucleotide Bookmark Sequences. Genes 2020, 11, 458.

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