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Open AccessConcept Paper

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

1
Clostridia Research Group, BBSRC/EPSRC Synthetic Biology Research Centre (SBRC), School of Life Sciences, Biodiscovery Institute, University of Nottingham, Nottingham NG7 2RD, UK
2
School of Life Sciences, Queen’s Medical Centre, Nottingham NG7 2UH, UK
3
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; https://doi.org/10.3390/genes11040458
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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