Yu Nie1, Peiqing Cong1, Xiaofeng Liu1, Min Wang1, Yaosheng Chen1, Zuyong He2. 1. Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China. 2. Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, 510006, People's Republic of China. zuyonghe@foxmail.com.
Abstract
OBJECTIVES: 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.
OBJECTIVES: 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.
Entities:
Keywords:
Bacterial-based negative selection system; CRISPR/Cas9; Cas nuclease; Single guide RNA; T7E1 assay