Park Rudolph V, Asbury Holly, Miller Stephen M
Department of Biological Sciences, University of Maryland, Baltimore County, Baltimore, MD 21250, USA.
MethodsX. 2020 Mar 10;7:100855. doi: 10.1016/j.mex.2020.100855. eCollection 2020.
A recently reported protocol demonstrates efficient CRISPR/Cas9 gene editing of [1]. The published protocol demonstrates transformation and editing of a wall-less strain of using plasmid encoded Cas9 and sgRNA. However, the published protocol utilizes a complex electroporation waveform that cannot be generated by most electroporation systems. It is unknown whether transformation via this complex electroporation waveform is essential for high efficiency of Cas9 edits, perhaps by optimizing Cas9 or guide RNA gene expression or incorporation into the genome. We demonstrate that a simple electroporation waveform can deliver plasmid encoded CRISPR/Cas9 into and edit the genome of a wall-less strain of as efficiently as the more complex waveform. Our modified electroporation protocol makes the plasmid based CRISPR/Cas9 genome editing method accessible to a greater number of researchers.•Our protocol uses a simple electroporation waveform to replace a complex waveform used to achieve efficient CRISPR/Cas9 gene editing in a wall-less strain of •We also increased concentration of plasmids to maintain high gene editing efficiency.•We minimized modifications to other steps of the original protocol.
最近报道的一项方案展示了对[1]的高效CRISPR/Cas9基因编辑。已发表的方案展示了使用质粒编码的Cas9和sgRNA对一种无壁菌株进行转化和编辑。然而，已发表的方案使用了一种大多数电穿孔系统无法生成的复杂电穿孔波形。通过这种复杂电穿孔波形进行转化对于Cas9编辑的高效率是否至关重要尚不清楚，这可能是通过优化Cas9或引导RNA基因表达或整合到基因组中实现的。我们证明，一种简单的电穿孔波形能够将质粒编码的CRISPR/Cas9导入一种无壁菌株的基因组并进行编辑，其效率与更复杂的波形相同。我们改进的电穿孔方案使更多的研究人员能够使用基于质粒的CRISPR/Cas9基因组编辑方法。•我们的方案使用简单的电穿孔波形替代用于在一种无壁菌株中实现高效CRISPR/Cas9基因编辑的复杂波形。•我们还提高了质粒浓度以维持高基因编辑效率。•我们尽量减少对原始方案其他步骤的修改。
