Assembling the Streptococcus thermophilus clustered regularly interspaced short palindromic repeats (CRISPR) array for multiplex DNA targeting.
In addition to the advantages of scalable, affordable, and easy to engineer, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) technology is superior for multiplex targeting, which is laborious and inconvenient when achieved by cloning multiple gRNA expressing cassettes. Here, we report a simple CRISPR array assembling method which will facilitate multiplex targeting usage. First, the Streptococcus thermophilus CRISPR3/Cas locus was cloned. Second, different CRISPR arrays were assembled with different crRNA spacers. Transformation assays using different Escherichia coli strains demonstrated efficient plasmid DNA targeting, and we achieved targeting efficiency up to 95% with an assembled CRISPR array with three crRNA spacers.
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Lijun
L
Guo
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Kun
K
Xu
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Zhiyuan
Z
Liu
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Adaptation and Evolution of Plateau Biota, Northwest Institute of Plateau Biology, Chinese Academy of Sciences, Xining, Qinghai 810008, China.
Cunfang
C
Zhang
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China.
Ying
Y
Xin
College of Animal Science & Technology, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: zhangzhy@nwsuaf.edu.cn.
Zhiying
Z
Zhang
D001483:Base Sequence; D064113:CRISPR-Cas Systems; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D018390:Gene Targeting; D010957:Plasmids / Q000235:genetics; D048249:Streptococcus thermophilus / Q000235:genetics*
D004247:DNA
CRISPR array; CRISPR/Cas; Cas9; Multiplex targeting; Streptococcus thermophilus