title: An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes.
abstract: Site-directed mutagenesis (SDM) methods are very important in modern molecular biology, biochemistry, and protein engineering. Here, we present a novel SDM method that can be used for multiple mutation generation using type IIs restriction enzymes. This approach is faster and more convenient than the overlap polymerase chain reaction (PCR) method due to its having fewer reaction steps and being cheaper than, but as convenient as, enzymatic assembly. We illustrate the usefulness of our method by introducing three mutations into the bacterial Streptococcus thermophilus Cas9 (bStCas9) gene, converting the humanized S. thermophilus Cas9 (hStCas9) gene into nuclease dead or H847A nickase mutants and generating sunnyTALEN mutagenesis from a wild-type TALEN backbone.
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Zhiqiang
authors: Z
authors: Zhang
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Kun
authors: K
authors: Xu
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors: Ying
authors: Y
authors: Xin
authors: College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: zhangzhy@nwsuaf.edu.cn.
authors: Zhiying
authors: Z
authors: Zhang
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D048249:Streptococcus thermophilus / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D015252:Deoxyribonucleases, Type II Site-Specific
keywords: Golden Gate cloning; SDM; bStCas9; hStCas9; sunnyTALEN