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.
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Acknowledgements
This work was jointly supported by National Transgenic Major Program (2016ZX08006003-006) and the Natural Science Foundation of Guangdong Province (2016A030313310).
Supporting information
Supplementary Table 1—sgRNA oligos for cloning into pX330-SP6.
Supplementary Table 2—sgRNA oligos for cloning into pX458.
Supplementary Table 3—Primers used for T7E1 assay.
Supplementary Table 4—Statistical analysis of colony formation in bacterial-based negative selection system.
Supplementary Fig. 1—Sequence analysis of cloned PCR products from cells transfected withCas9/single guide RNA (sgRNA) expression plasmids.
Supplementary Fig. 2—Correlation between insertions or deletions (indel) rate reflected by TA-cloning sequencing results and bacterial survival rate.
Supplementary Fig. 3—Transformation results of the bacterial-based negative selection assay.
Supplementary Fig. 4—Leaky expression of EGFP in E. coli DH5α cells under the control of SP6 promoter.
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Nie, Y., Cong, P., Liu, X. et al. Development of a bacterial-based negative selection system for rapid screening of active single guide RNAs. Biotechnol Lett 39, 351–358 (2017). https://doi.org/10.1007/s10529-016-2259-0
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DOI: https://doi.org/10.1007/s10529-016-2259-0