Comparison of the Off-Target Effects Among One-Base to Three-Base Mismatched Targets of gRNA Using a Blue to White Assay

: The present study was designed to test a new strategy for comparing the off-target effects of CRISPR/cas9 employing a blue/white colony based assay. Eight types of AmpR plasmids with matched, one base mismatched, two bases mismatched and three bases mismatched targets were constructed. The wild typed alpha peptide of the beta-glactosidase gene and some with mutations, chosen as the targets, were successfully subcloned into the plasmids in an inframe pattern. The relevant gRNA and cas9 were subcloned into the ChloR plasmid. Transformation of the plasmid harboring the target gene yielded blue colonies, while white colonies formed in the plates following the co-transformation of target gene plasmid and the CRISPR/cas9 plasmid. Colonies in different levels of blue color were formed from the plasmids having completely matched target and mismatched targets. The appearance of different blue levels following the co-transformation of the different target plasmid and CRISPR/cas9 plasmid qualitatively indicated the effectiveness of the gRNA, while the ratio of the AMP/CL colonies can be used for relatively quantitative evaluation. The target and off-target effects among different targets of gRNA were differentially identifiable and these effects in turn demonstrated the effectiveness of the blue to white assay. Either the qualitative or the quantitative analysis of the blue to white depends on the final interactions among the target, the cas9 and the gRNA. Clearly, when co-transformation is replaced by single transformation using a specialized nanoparticle containing these three components, the target and off-target effect will be more accurately evaluated. Thus, this study provided a model for application of nanoparticle in gene editing studies.