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

Rowe, Peter; Bolt, Edward L.; Humphreys, Christopher M.; Minton, Nigel P.

doi:10.3390/genes11040458

Authors

Seys

Peter Rowe

ED BOLT ED.BOLT@NOTTINGHAM.AC.UK
Associate Professor

CHRISTOPHER HUMPHREYS C.HUMPHREYS@NOTTINGHAM.AC.UK
Senior Research Fellow

NIGEL MINTON nigel.minton@nottingham.ac.uk
Professor of Applied Molecular Microbiology

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.

Citation

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. https://doi.org/10.3390/genes11040458

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
DOI	https://doi.org/10.3390/genes11040458
Keywords	bookmark; CRISPR/Cas9; complementation; Clostridium; knock-out
Public URL	https://nottingham-repository.worktribe.com/output/4336552
Publisher URL	https://www.mdpi.com/2073-4425/11/4/458