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- Title
A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method.
- Authors
Dong, Mengjie; Wang, Fei; Li, Qingqing; Han, Rui; Li, Aitao; Zhai, Chao; Ma, Lixin
- Abstract
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.
- Subjects
SITE-specific mutagenesis; SINGLE-stranded DNA; DIGESTION; MUTAGENESIS; EXONUCLEASES
- Publication
Applied Microbiology & Biotechnology, 2020, Vol 104, Issue 9, p3993
- ISSN
0175-7598
- Publication type
Article
- DOI
10.1007/s00253-020-10477-3