An efficient method for multiple site-directed mutagenesis using type IIs restriction enzymes

作者：Zhang, Zhiqiang; Xu, Kun; Xin, Ying; Zhang, Zhiying^*

来源：Analytical Biochemistry, 2015, 476: 26-28.

DOI：10.1016/j.ab.2015.01.010

• SDM
• Golden Gate cloning
• bStCas9
• hStCas9
• sunnyTALEN

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 us 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.

• 出版日期2015-5-1
• 单位西北农林科技大学