Preprint Concept Paper Version 1 Preserved in Portico This version is not peer-reviewed

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

Version 1 : Received: 20 March 2020 / Approved: 23 March 2020 / Online: 23 March 2020 (00:50:23 CET)

A peer-reviewed article of this Preprint also exists.

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. 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.

Journal reference: Genes 2020, 11, 458
DOI: 10.3390/genes11040458

Abstract

Phenotypic complementation of gene knock-outs 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, 9 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 incorporation of ‘watermark’ sequences into the complementing genes.

Subject Areas

bookmark; CRISPR/Cas9; complementation; clostridium; knock-out

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