@article { , title = {A gold standard, CRISPR/Cas9-based complementation strategy reliant on 24 nucleotide bookmark sequences}, 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.}, doi = {10.3390/genes11040458}, eissn = {2073-4425}, issn = {2073-4425}, issue = {4}, journal = {Genes}, publicationstatus = {Published}, publisher = {MDPI}, url = {https://nottingham-repository.worktribe.com/output/4336552}, volume = {11}, keyword = { bookmark, CRISPR/Cas9, complementation, Clostridium, knock-out}, year = {2020}, author = {Seys, François M. and Rowe, Peter and Bolt, Edward L. and Humphreys, Christopher M. and Minton, Nigel P.} }