Seys François M, Rowe Peter, Bolt Edward L, Humphreys Christopher M, Minton Nigel P
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.
Genes (Basel). 2020 Apr 23;11(4):458. doi: 10.3390/genes11040458.
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 CRISPR/Cas9 but could be designed for any CRISPR/Cas system. For proof of concept, nine bookmarks were tested in . 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.
基因敲除的表型互补是确定功能的关键步骤。在此，我们描述了一种用于“金标准”互补的简单策略，即在利用CRISPR/Cas9的过程中，将突变等位基因原位替换为野生型（WT）等位基因。该方法依赖于事先将一个独特的24个核苷酸（nt）的“书签”序列整合到突变等位基因中，以便在其由Cas9介导被WT等位基因替换的过程中作为引导RNA的靶标。该书签由一个23 nt的Cas9靶标序列加上一个额外的nt组成，以确保缺失是框内的。在此，书签是针对CRISPR/Cas9量身定制的，但也可为任何CRISPR/Cas系统设计。为了验证概念，在……中测试了9个书签。互补效率达到了91%。由于互补菌株与其亲本无法区分，通过将“水印”序列整合到互补基因中，可能消除对污染的担忧。
