State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, School of Life Sciences, Hubei University, Wuhan, People's Republic of China.
Appl Microbiol Biotechnol. 2020 May;104(9):3993-4003. doi: 10.1007/s00253-020-10477-3. Epub 2020 Mar 9.
A PCR-independent in vitro site-directed mutagenesis method was established. Cas12a from Francisella novicida (FnCas12a) linearizes the plasmid with single digestion. T5 exonuclease removes the target nucleotide. A short single- or double-stranded mutagenic oligonucleotide introduces the mutation. This rapid and simple mutagenesis method is referred to as FnCas12a and T5 exonuclease mediated site-directed mutagenesis system (CT5-SDM). The platform is also suitable for the mutagenesis of plasmids larger than 10 kb. KEY POINTS: Site-directed mutagenesis mediated by single-stranded DNA. Removing target site with T5 exonuclease. Highly efficient cleavage of target DNA with FnCas12a.
建立了一种不依赖于 PCR 的体外定点突变方法。来自弗朗西斯氏菌属 novicida（FnCas12a）的 Cas12a 对质粒进行单次消化使其线性化。T5 外切酶切除靶核苷酸。短的单链或双链突变寡核苷酸引入突变。这种快速简单的诱变方法称为 FnCas12a 和 T5 外切酶介导的定点突变系统（CT5-SDM）。该平台也适用于大于 10kb 的质粒的诱变。要点：单链 DNA 介导的定点突变。T5 外切酶去除靶位点。FnCas12a 高效切割靶 DNA。
