title: Development of a bacterial-based negative selection system for rapid screening of active single guide RNAs.
abstract: OBJECTIVE
  To develop an in vitro method for rapid evaluation of the capability of a designed single guide RNAs (sgRNAs) to guide Cas9 nucleases to cleave target loci in mammalian cells.
  
  
  RESULTS
  We constructed a Cas9/sgRNA plasmid with two SP6 promoters to simultaneously express Cas9 nuclease and the sgRNA and a negative selection plasmid harbouring a target site of the sgRNA. After co-transforming chemically competent E. coli DH5α cells with the two plasmids, the transformants were plated at a low density on two LB plates: one containing only ampicillin and the other containing both ampicillin and chloramphenicol. The colony-count on the ampicillin + chloramphenicol plate was compared with that on the ampicillin-only plate to calculate the survival percentage. The survival % was negatively correlated with the genome editing efficiency of the sgRNA in mammalian cells evaluated by a T7 endonuclease 1 (T7E1) assay (r ranged from -0.8 to -0.92). This system eliminates the need for cell culture, transfection, FACS sorting, PCR and T7E1 nuclease treatment, and significantly reduces the cost of screening for active sgRNAs, especially in the case of large-scale screening.
  
  
  CONCLUSIONS
  We have developed a bacterial-based negative selection system for rapid screening of active sgRNAs in mammalian cells at a very low cost.
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Nie
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Peiqing
authors.initials: P
authors.lastname: Cong
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Liu
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Wang
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China.
authors.forename: Yaosheng
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China. zuyonghe@foxmail.com.
authors.forename: Zuyong
authors.initials: Z
authors.lastname: He
mesh_terms: D000818:Animals; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D005820:Genetic Testing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D054643:INDEL Mutation / Q000235:genetics; D051379:Mice; D010957:Plasmids / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics* / Q000378:metabolism; D034421:Sus scrofa
chemical_list: D000094704:RNA, Guide, CRISPR-Cas Systems
keywords: Bacterial-based negative selection system; CRISPR/Cas9; Cas nuclease; Single guide RNA; T7E1 assay