A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method.
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.
['Dong|Mengjie|M|', 'Wang|Fei|F|', 'Li|Qingqing|Q|', 'Han|Rui|R|', 'Li|Aitao|A|', 'Zhai|Chao|C|', 'Ma|Lixin|L|']
D004277:DNA, Single-Stranded D004926:Escherichia coli D005603:Francisella D016297:Mutagenesis, Site-Directed D009841:Oligonucleotides D016133:Polymerase Chain Reaction
Single digestion Single-stranded oligonucleotide Site-directed mutagenesis FnCas12a T5 exonuclease