A single digestion, single-stranded oligonucleotide mediated PCR-independent site-directed mutagenesis method

Applied Microbiology and Biotechnology
Mengjie DongLixin Ma

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.

References

Jun 7, 2001·Proceedings of the National Academy of Sciences of the United States of America·H M EllisD L Court
Aug 12, 2004·Nucleic Acids Research·Lei ZhengJean-Louis Reymond
Feb 13, 2007·Nature Methods·Mamie Z Li, Stephen J Elledge
Jan 31, 2009·Nature Protocols·Shyam K SharanDonald L Court
Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Jan 9, 2010·Science·Philippe Horvath, Rodolphe Barrangou
Jan 14, 2012·Nucleic Acids Research·Yongwei ZhangWinfried Edelmann
Feb 18, 2012·Nature·Blake WiedenheftJennifer A Doudna
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Sep 6, 2012·Proceedings of the National Academy of Sciences of the United States of America·Giedrius GasiunasVirginijus Siksnys
Jan 22, 2013·International Journal of Medical Microbiology : IJMM·Eva SchunderKlaus Heuner
Mar 19, 2013·Annual Review of Biochemistry·Rotem SorekBlake Wiedenheft
Jun 9, 2016·Nature Biotechnology·Yongsub KimYoung Hoon Sung
Jun 9, 2016·Nature Structural & Molecular Biology·Faizah A AlMalkiPeter J Artymiuk
Jun 14, 2016·ACS Synthetic Biology·Shi-Yuan LiJin Wang
Nov 7, 2017·Nucleic Acids Research·Michal A SwiatPascale Daran-Lapujade

❮ Previous
Next ❯

Citations


❮ Previous
Next ❯

Datasets Mentioned

BETA
MK673548.1
KU998257.1
LC466107.1
MN887111

Methods Mentioned

BETA
affinity purification
electrophoresis
PCR

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.