期刊
ANALYTICAL BIOCHEMISTRY
卷 476, 期 -, 页码 26-28出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2015.01.010
关键词
SDM; Golden Gate cloning; bStCas9; hStCas9; sunnyTALEN
资金
- National Nature Science Foundation of China (NSFC) [31172186]
- Ministry of Agriculture of China (948 Program) [2013-Z27]
- Ministry of Science and Technology of China (973 Program) [2011CBA01002]
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. (C) 2015 Elsevier Inc. All rights reserved.
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