A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method
- 1 May 2020
- journal article
- research article
- Published by Springer Nature in Applied Microbiology and Biotechnology
- Vol. 104 (9) , 3993-4003
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.Keywords
This publication has 30 references indexed in Scilit:
- CRISPR-Mediated Adaptive Immune Systems in Bacteria and ArchaeaAnnual Review of Biochemistry, 2013
- Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteriaProceedings of the National Academy of Sciences, 2012
- A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial ImmunityScience, 2012
- RNA-guided genetic silencing systems in bacteria and archaeaNature, 2012
- SLiCE: a novel bacterial cell extract-based DNA cloning methodNucleic Acids Research, 2012
- CRISPR/Cas, the Immune System of Bacteria and ArchaeaScience, 2010
- Recombineering: a homologous recombination-based method of genetic engineeringNature Protocols, 2009
- An efficient one-step site-directed and site-saturation mutagenesis protocolNucleic Acids Research, 2004
- A helical arch allowing single-stranded DNA to thread through T5 5'-exonucleaseNature, 1996
- Site-directed mutagenesis by overlap extension using the polymerase chain reactionGene, 1989